Method for generating genetically altered antigens

ABSTRACT

Dominant negative alleles of human mismatch repair genes can be used to generate hypermutable cells and organisms. By introducing these genes into cells and transgenic animals, new cell lines and animal varieties with novel and useful properties can be prepared more efficiently than by relying on the natural rate of mutation. These methods are useful for generating genetic diversity within genes encoding for therapeutic antigens to produce altered polypeptides with enhanced antigenic and immunogenic activity. Moreover, these methods are useful for generating effective vaccines.

TECHNICAL FIELD OF THE INVENTION

[0001] The invention is related to the area of genetic alterations of antigens as potent vaccines. In particular it is related to the field of mutagenesis.

BACKGROUND OF THE INVENTION

[0002] The use of vaccines to build immunity against foreign and/or endogenous polypeptides provides an effective and selective strategy for treating the underlying cause of disease. In particular is the use of killed viruses such as the polio mellitus and the Hepatitis B virus (John T. J. (2000) New Engl. J. Med. 14:806-807). Standard methods for generating vaccines against candidate pathogenic organisms or molecules are known by those skilled in the art. Vaccines for human use are developed in animal models to survey for the ability of killed or defective whole agents such as parasites, viruses or recombinant polypeptides to cause immunity against infection of the pathogenic agent (Boyce T. G. et al. (2000) Vaccine 19:217-226). Briefly, rodents such as mice or rats are injected with a purified antigen in the presence of adjuvant to generate an immune response (Boyce T. G. et al. (2000) Vaccine 19:217-226). Unfortunately, not all antigens are capable of eliciting a strong immune response when injected into a host organism (Hoshino Y. and A. Z. Kapikian (2000) J. Health Popul. Nutr. 18:5-14; Orenstein W. A. et al. (2000) Am. J. Public Health 90:1521-525; Lechmann M. and T. J. Liang (2000) Semin. Liver Dis. 20:211-226). While the reasons for the lack of immune response are not clear, some factors, such as the lack of T-cell epitopes which are important for stimulating cellular-mediated immune responses, may be absent within a given antigen (Ausiello C. M. et al. (1999) Infect. Immun.67:40644071; Brosstoff S. (1995) Adv. Exp. Med. Biol. 383:249-254). In the case of parasitic infections, the development of effective vaccines has been hampered by the presence of many different developmental stages that occur within an infected host and that a diverse array of allelic forms occurs within genes encoding for prominent surface antigens (MALARIA OBSTACLES AND OPPORTUNITIES, Oaks, S. C. et al., Eds., National Academy Press, p 1, 1991; Anders, R. F. “Vaccines Against Asexual Blood Stages of Plasmodium falciparum” NEW GENERATION VACCINES, 2^(nd) Ed., Anders, R. F., pp. 1035-1055, 1997). It is believed by many skilled in the art that the generation of highly antigenic polypeptides may overcome these limitations and produce a protective immune response to pathogens (McLeod R. et al. (1995) Curr. Opin. Immunol.7:539-552).

[0003] A method for generating diverse sequences within a polypeptide would be useful for the creation of more potent therapeutic agents. Moreover, the generation of randomly altered nucleotides and encoded polypeptide residues throughout an entire antigen molecule may result in new reagents that are: 1) more antigenic; 2) more immunogenic; and 3) have beneficial pharmacokinetic properties.

SUMMARY OF THE INVENTION

[0004] The invention described herein is directed to the use of random genetic mutation of a polypeptide in vivo by blocking the endogenous mismatch repair (MMR) activity of a host cell yielding structurally altered antigens that can be screened for antigenicity and immunogenicity in comparison to the wild type molecule. The use of mammalian cell-based high throughput screens as taught by this application will facilitate identification of randomly altered antigens that may serve as effective vaccines. Moreover, the invention describes methods for repeated in vivo genetic alterations and selection for antigens with enhanced immunogenicity and pharmacokinetic profiles.

[0005] The ability to develop and screen genetically altered mammalian cells that secrete structurally altered polypeptides in a high throughput manner provides a valuable method for creating vaccines for therapeutic development. A potential problem in generating potent vaccine antigens against endogenous to the mammalian host is the source of antigen production. In many instances recombinant polypeptides that are naturally produced by mammalian cells are generated recombinantly using insect, yeast or bacterial expression systems. These sources typically produce large amounts of proteins that are distinct from the mammalian-produced polypeptides, and may differ from the natural protein due to altered folding or altered post-translational modifications such as hyperglycosylation. The invention described herein is directed to the creation of genetically altered mammalian cell hosts that produce structurally altered polypeptides as vaccine agents via the blockade of MMR.

[0006] The present invention facilitates the generation of highly antigenic polypeptides as vaccines. The advantages of the present invention are further described in the examples and figures described herein.

[0007] The present invention provides methods for generating genetically altered antigens in vivo, whereby the antigen possesses desired biochemical property(s), such as, but not limited to, increased antigenicity and immunogenicity. One method for identifying antigens with increased antigenicity is through the screening of mismatch repair (“MMR”) defective cell clones that produce desired antigens.

[0008] The invention also provides methods for rendering cells expressing a target antigen hypermutable. The cells include, but are not limited to rodent, primate, human, plant, yeast or bacterial cells. The antigens can be generated from endogenous genes or from introduced transgenes.

[0009] The invention also provides methods for generating genetically altered cell lines that express antigenic polypeptides.

[0010] In some embodiments, the invention provides methods for generating genetically altered cell lines that produce immunogenic polypeptides.

[0011] In other embodiments, the invention provides methods for producing an antigen expression cassette for high throughput screening of altered polypeptides in vivo.

[0012] In other embodiments, the invention provides methods of mutating a gene of interest in a mismatch repair defective cell.

[0013] In some embodiments, the invention provides methods of creating genetically altered antigens in vivo by blocking the MMR activity of the cell host.

[0014] Still other embodiments of the invention provide methods of creating genetically altered polypeptides in vivo by transfecting genes encoding for an antigen in a MMR defective cell host.

[0015] The invention also embraces methods of creating antigens with increased immunogencity due to genetic alterations within the antigen-encoding gene by blocking endogenous MMR of the cell host.

[0016] In some embodiments, the invention provides methods of creating a library of randomly altered antigens from mammalian cells by blockade of MMR of the cell host.

[0017] In other embodiments, the invention provides methods of creating antigens with enhanced pharmacokinetic profiles due to genetic changes within the encoding gene by blocking endogenous MMR of the cell host.

[0018] The invention also provides methods of creating genetically altered antigens in MMR defective cells as vaccine agents.

[0019] In some embodiments, the invention provides methods for high throughput screening of antigens produced by MMR defective cells.

[0020] These and other objects of the invention are provided by one or more of the embodiments described below. In one embodiment of the invention, a method for making MMR defective cell lines expressing a target antigen will be provided. A polynucleotide encoding a dominant negative allele of an MMR gene is introduced into a target antigen-producing cell. The cell becomes hypermutable as a result of the introduction of the gene.

[0021] In another embodiment of the invention, an isolated hypermutable cell producing antigenic peptides is provided. The cell is defective for mismatch repair and exhibits an enhanced rate of hypermutation. The cell produces a polypeptide from a mutated gene encoding for the polypeptide.

[0022] In another embodiment of the invention, a method is provided for introducing a mutation into an endogenous gene encoding for a target polypeptide. A polynucleotide encoding a dominant negative allele of a MMR gene is introduced into a cell. The cell becomes hypermutable as a result of the introduction and expression of the MMR gene allele. The cell further comprises a gene of interest. The cell is grown and tested to determine whether the gene encoding for a polypeptide of interest harbors a mutation.

[0023] In another embodiment of the invention, a method is provided for producing a cell-based screening assay to identify antigenic proteins as vaccines. A polynucleotide encoding a dominant negative allele of a MMR gene is introduced into a cell expressing a secreted antigen. The cell becomes hypermutable as a result of the introduction of the gene. The cell is grown and conditioned medium from the cell is tested for the expression of antigenic polypeptides.

[0024] In another embodiment of the invention, a gene, or set of genes encoding for polypeptides or a combination therein, are introduced into a mammalian cell host that is defective in MMR. The cell is grown and clones are analyzed for antigens with enhanced antigenicity.

[0025] In another embodiment of the invention, a method is provided for producing a cell-based screening assay to identify antigenic proteins as vaccines. A polynucleotide encoding a secreted antigen is introduced into a naturally MMR defective cell. The gene is hypermutable as a result of the introduction of MMR deficiency. The cell is grown and conditioned medium from the cell is tested for the expression of antigenic polypeptides.

[0026] In another embodiment of the invention, a method will be provided for restoring genetic stability in a cell containing a polynucleotide encoding for a dominant negative allele of a MMR gene. The expression of the dominant negative MMR gene is suppressed and the cell restores its genetic stability including but not limited to genetic stability within the antigen-encoding genes.

[0027] In another embodiment of the invention, a method will be provided for restoring genetic stability in a cell containing a polynucleotide encoding a dominant negative allele of an MMR gene and a newly selected phenotype. The expression of the dominant negative mismatch repair gene is suppressed and the cell restores its genetic stability and the new phenotype is stable.

[0028] These and other embodiments of the invention provide the art with methods that can generate enhanced mutability in cells and animals as well as providing cells and animals harboring potentially useful mutations for the large-scale production of highly antigenic polypeptides as potent vaccines.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029]FIG. 1: In situ β-galactosidase staining of TKPMS134/pCAR-OF or TKvect/pCAR-OF cells to assay for MMR defective cells containing genetically altered β-galactosidase genes. Arrows indicate Blue (β-galactosidase positive) cells.

[0030]FIG. 2: Schematic representation of sequence alterations of the β-galactosidase gene produced by MMR defective host cells

[0031]FIG. 3: Schematic representation of sec-hist secretion proteins for screening of structurally altered antigenic polypeptides and the sec-hist expression cassette (SEQ ID NO:17). Panel A: Schematic representation of sec-hist protein; Panel B: Sequence of sec-hist expression cassette. In Panel B, the italic bold sequence represents a HindIII site for subcloning; the double underlined sequence on the 5′ end represents leader sequence from the human IL-2; the underlined sequence on the 3′ end represents the poly histidine sequence followed by 2 termination codons; sequence in capital letters represents sequence from the polylinker region of pUC 18; the polylinker contains the following restriction enzymes for cloning cDNAs: SacI-SacII-NotI-XbaI-SpeI-BamHI-SmaI-PstI-EcoRI.

[0032]FIG. 4: Schematic diagram for high throughput screening of conditioned medium from TK clones for the identification of antigenic sec-hist polypeptides. Assays employ an in vitro antigenicity test using splenocytes from naive mice (non-primed) and antigen-exposed (primed) mice. Clones exhibiting positive CM are then genetically analyzed to confirm structural alterations within the sec-hist sequence, followed by protein purification and retesting of purified proteins. Purified proteins with the best stimulatory activity are then screened in vivo for immunogenicity. The screening assay can be repeated for several rounds to add additional alterations within the antigen (long arrow).

[0033] The inventors have discovered a method for developing hypermutable cells producing therapeutic antigens by taking advantage of the conserved mismatch repair (MMR) process of host cells. Dominant negative alleles of such genes, when introduced into cells or transgenic animals, increase the rate of spontaneous mutations by reducing the effectiveness of DNA repair and thereby render the cells or animals hypermutable. Hypermutable cells or animals can then be utilized to develop new mutations in a gene or genes of interest. Blocking MMR in cells producing antigens (including, but not limited to, mammalian cells, plant cells, yeast cells, and prokaryotic cells) can enhance the rate of mutation within the gene encoding for the antigen that can be screened to identify clones producing structurally altered polypeptides with enhanced antigenicitiy and immunogenicity.

[0034] In one aspect of the invention, the methods are useful for the production of antigens that have increased antigenicity and/or immunogenicity. Such antigens may be used as immunogens to elicit immune responses in animals against these antigens.

[0035] The antigens may be derived from, for example, pathogenic organisms or cancer cells such that an immune response is directed against the pathogenic organism or cancer cell and exerts an effect on the organism or cancer cell. The effect may be, for example, to prevent, inhibit or terminate the growth of the pathogenic organism or cancer cell when an immunogenic amount of the antigen is administered to an animal.

[0036] The pathogenic organisms-from which antigens may be derived include bacteria, fungi, parasitic protozoa, helminths, and viruses. Non-limiting examples include species of the following genera: Staphylococcus, Streptococcus, Bacillus, Bordetella, Clostridium, Escherichia, Haemophilus, Helicobacter, Klebsiella, Listeria, Salmonella, Vibrio, Yersinia, Neisseria, Treponema, Borrelia, Corynebacterium, Mycobacterium, Mycoplasma, Chlamydia, Acremonium, Aspergillus, Blastomyces, Candida, Acanthamoeba, Ascaris, Babesia, Cryptosporidium, Echinococcus, Entamoeba, Giardia, Necator, Ancylostoma, Unicinaria, Leishmania, Onchocerca, Plasmodium, Schistosoma, Strongyloides, Taenia, Toxoplasma, Trichinella, Trichomonas, Trichuris, Trypanosoma, Dirofilaria, Brugia, Wuchereria, and Eimeria. Non-limiting examples of viruses include adenovirus, arborviruses, coronavirus, cytomegalovirus, enteroviruses, Epstein-Barr virus, hepatitis viruses, herpes viruses, immunodeficiency viruses (e.g., HIV, FIV SIV), papilloma viruses, T-cell leukemia viruses, influenza viruses, mumps viruses, parainfluenzae viruses, parvoviruses, poxviruses, Rabies virus, respiratory syncytial virus, rhinoviruses, rotaviruses, Rubella viruses, and varicella-zoster viruses.

[0037] The antigens derived from the pathogenic organisms, for example, may be antigens known to elicit an immune response, for which an enhanced immune response is desired, or the antigen may be one that is known to generate a weak response for which an enhanced response is desired. It is also possible that some antigens that did not previously elicit an immune response will become antigenic as a result of the methods of the invention and the phenomenon of hypermutability of cells which contain dominant negative alleles of mismatch repair genes.

[0038] The antigens produced by the method of the invention are novel immunogens that may be administered in an appropriate pharmaceutical carrier, such as an adjuvant, for administration to animals as a vaccine. The antigens of the invention may be administered to animals in immunogenic amounts such that an antibody and/or a cell-mediated immune response is elicited. The administration of the antigens of the invention may be administered as a single dose, or, preferably as a plurality of doses to effect a boosted immune response. The route of administration may be any accepted route of immunization including, for example, oral, intrmuscular, intrperitoneal, subcutaneous, intradermal, intranasal, or transdermal.

[0039] Doses for humans can readily be extrapolated from animal studies as taught by Katocs et al., Chapter 27 of REMINGTON'S PHARMACEUTICAL SCIENCES, 18^(th) Edition, Gennaro (Ed.) Mack Publishing Co., Easton, Pa., 1990. Immunogenic dosages can be adjusted by one skilled in the art, and may vary depending on several factors, including the age, health, physical condition, weight, type and extent of the disease or disorder of the recipient, frequency of treatment, the nature of concurrent therapy, if required, and the nature and scope of the desired effect(s) (Nies et al., Chapter 3, GOODMAN & GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 9th Ed., Hardman et al., Eds., McGraw-Hill, New York, N.Y., 1996). Typically, an immunogenic amount of the antigens of the invention will be in the range of about 5 to about 100 μg.

[0040] The antigens of the present invention may be administered as single antigens or may be administered as combinations of antigens. As a non-limiting example, the antigen combinations may be antigens of the same pathogenic organism, or may be antigens of different pathogenic organisms, such that immune responses are elicited to more than one pathogenic organism.

[0041] The antigens of the present invention are hypermutated by the methods of the invention which take advantage of the mismatch repair system. The process of MMR, also called mismatch proofreading, is carried out by protein complexes in cells ranging from bacteria to mammalian cells. A MMR gene is a gene that encodes for one of the proteins of such a mismatch repair complex. Although not wanting to be bound by any particular theory of mechanism of action, a MMR complex is believed to detect distortions of the DNA helix resulting from non-complementary pairing of nucleotide bases. The non-complementary base on the newer DNA strand is excised, and the excised base is replaced with the appropriate base, which is complementary to the older DNA strand. In this way, cells eliminate many mutations that occur as a result of mistakes in DNA replication.

[0042] Dominant negative alleles cause a MMR defective phenotype even in the presence of a wild-type allele in the same cell. An example of a dominant negative allele of a MMR gene is the human gene hPMS2-134, which carries a truncating mutation at codon 134. The mutation causes the product of this gene to abnormally terminate at the position of the 134th amino acid, resulting in a shortened polypeptide containing the N-terminal 133 amino acids. Such a mutation causes an increase in the rate of mutations, which accumulate in cells after DNA replication. Expression of a dominant negative allele of a mismatch repair gene results in impairment of mismatch repair activity, even in the presence of the wild-type allele. Any allele that produces such effect can be used in this invention.

[0043] Dominant negative alleles of a MMR gene can be obtained from the cells of humans, animals, yeast, bacteria, or other organisms (Prolla T. A. et al. (1994) Science 264:1091-1093; Strand M. et al. (1993) Nature 365:274-276; Su, S. S. et al. (1988) J. Biol. Chem. 263:6829-6835). Such alleles can be identified by screening cells for defective MMR activity. Cells from animals or humans with cancer can be screened for defective mismatch repair. Cells from colon cancer patients may be particularly useful. Genomic DNA, cDNA, or mRNA from any cell encoding a MMR protein can be analyzed for variations from the wild type sequence. Dominant negative alleles of a MMR gene can also be created artificially, for example, by producing variants of the hPMS2-134 allele or other MMR genes. Various techniques of site-directed mutagenesis can be used. The suitability of such alleles, whether natural or artificial, for use in generating hypermutable cells or animals can be evaluated by testing the mismatch repair activity caused by the allele in the presence of one or more wild-type alleles, to determine if it is a dominant negative allele.

[0044] A cell or an animal into which a dominant negative allele of a mismatch repair gene has been introduced will become hypermutable. This means that the spontaneous mutation rate of such cells or animals is elevated compared to cells or animals without such alleles. The degree of elevation of the spontaneous mutation rate can be at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1000-fold that of the normal cell or animal. The use of chemical mutagens such as but limited to methane sulfonate, dimethyl sulfonate, O6-methyl benzadine, MNU, ENU, etc. can be used in MMR defective cells to increase the rates an additional 10 to 100 fold that of the MMR deficiency itself.

[0045] According to one aspect of the invention, a polynucleotide encoding for a dominant negative form of a MMR protein is introduced into a cell. The gene can be any dominant negative allele encoding a protein, which is part of a MMR complex, for example, PMS2, PMS1, MLH1, or MSH2. The dominant negative allele can be naturally occurring or made in the laboratory. The polynucleotide can be in the form of genomic DNA, cDNA, RNA, or a chemically synthesized polynucleotide.

[0046] The polynucleotide can be cloned into an expression vector containing a constitutively active promoter segment [such as but not limited to CMV, SV40, Elongation Factor (EF) or LTR sequences] or to inducible promoter sequences such as the steroid inducible pIND vector (InVitrogen), tetracycline, or MMTV, where the expression of the dominant negative MMR gene can be regulated. The polynucleotide can be introduced into the cell by transfection.

[0047] According to another aspect of the invention, a gene, a set of genes or a chimeric gene encoding for whole or parts of a therapeutic antigen can be transfected into MMR deficient cell hosts, the cell is grown and screened for clones containing genetically altered genes encoding for antigens with new biochemical features including but not limited to increased antigenicity. MMR defective cells may be of human, primates, mammals, rodent, plant, yeast or of the prokaryotic kingdom.

[0048] Transfection is any process whereby a polynucleotide is introduced into a cell. The process of transfection can be carried out in a living animal, e.g., using a vector for gene therapy, or it can be carried out in vitro, e.g., using a suspension of one or more isolated cells in culture. The cell can be any type of eukaryotic cell, including, for example, cells isolated from humans or other primates, mammals or other vertebrates, invertebrates, and single celled organisms such as protozoa, yeast, or bacteria.

[0049] In general, transfection will be carried out using a suspension of cells, or a single cell, but other methods can also be applied as long as a sufficient fraction of the treated cells or tissue incorporates the polynucleotide so as to allow transfected cells to be grown and utilized. The protein product of the polynucleotide may be transiently or stably expressed in the cell. Techniques for transfection are well known. Available techniques for introducing polynucleotides include but are not limited to electroporation, transduction, cell fusion, the use of calcium chloride, and packaging of the polynucleotide together with lipid for fusion with the cells of interest. Once a cell has been transfected with the dominant negative MMR gene, the cell can be grown and reproduced in culture. If the transfection is stable, such that the gene is expressed at a consistent level for many cell generations, then a cell line results.

[0050] An isolated cell is a cell obtained from a tissue of humans or animals by mechanically separating out individual cells and transferring them to a suitable cell culture medium, either with or without pretreatment of the tissue with enzymes, e.g., collagenase or trypsin. Such isolated cells are typically cultured in the absence of other types of cells. Cells selected for the introduction of a dominant negative allele of a mismatch repair gene may be derived from a eukaryotic organism in the form of a primary cell culture or an immortalized cell line, or may be derived from suspensions of single-celled organisms.

[0051] A polynucleotide encoding for a dominant negative form of a MMR protein can be introduced into the genome of an animal by producing a transgenic animal. The animal can be any species for which suitable techniques are available to produce transgenic animals. For example, transgenic animals can be prepared from domestic livestock, e.g., bovine, swine, sheep, goats, horses, etc.; from animals used for the production of recombinant proteins, e.g., bovine, swine, or goats that express a recombinant polypeptide in their milk; or experimental animals for research or product testing, e.g., mice, rats, guinea pigs, hamsters, rabbits, etc. Cell lines that are determined to be MMR defective can then be used as a source for producing genetically altered genes encoding for therapeutic antigens in vitro by introducing whole, intact genes and/or chimeric genes encoding for a therapeutic antigen(s) into MMR defective cells from any tissue of the MMR defective animal.

[0052] Once a transfected cell line or a colony of transgenic animals has been produced, it can be used to generate new mutations in one or more gene(s) of interest. A gene of interest can be any gene naturally possessed by the cell line or transgenic animal or introduced into the cell line or transgenic animal. An advantage of using such cells or animals to induce mutations is that the cell or animal need not be exposed to mutagenic chemicals or radiation, which may have secondary harmful effects, both on the object of the exposure and on the workers. However, chemical mutagens may be used in combination with MMR deficiency, which renders such mutagens less toxic due to an undetermined mechanism. Hypermutable animals can then be bred and selected for those producing genetically variable cells that may be isolated and cloned to identify new cell lines that are useful for producing structurally altered polypeptides. Once an altered polypeptide is identified, the dominant negative MMR gene allele can be removed by directly knocking out the allele by technologies used by those skilled in the art or by breeding to mates lacking the dominant negative allele to select for offspring with a desired trait and a stable genome. Another alternative is to use a CRE-LOX expression system, whereby the dominant negative allele is spliced from the animal genome once an animal containing a genetically diverse protein profile has been established. Yet another alternative is the use of inducible vectors such as the steroid induced pIND (InVitrogen) or pMAM (Clonetech) vectors which express exogenous genes in the presence of corticosteroids.

[0053] Mutations can be detected by analyzing for alterations in the genotype of the cells or animals, for example by examining the sequence of genomic DNA, cDNA, messenger RNA, or amino acids associated with the gene of interest. Mutations can also be detected by screening for the production of antigenicity. A mutant polypeptide can be detected by identifying alterations in electrophoretic mobility, spectroscopic properties, or other physical or structural characteristics of a protein encoded by a mutant gene. One can also screen for altered function of the protein in situ, in isolated form, or in model systems. One can screen for alteration of any property of the cell or animal associated with the function of the gene of interest, such as but not limited to antigenicity.

[0054] According to another aspect of the invention, a high throughput mammalian cell-based assay is presented. A MMR defective cell line is transfected with a secretion cassette containing a leader sequence for secretion at the N-terminus fused to the target antigen. Cells are grown and clones are plated by limiting dilution into microtitre plates and conditioned medium are screened for antigenic peptides. The advantage of such an approach is that the antigen is more similar to the natural polypeptide than it would be if produced by bacterial, yeast or baculovirus systems which tend to cause misfolding and/or distorted post-translational modifications.

[0055] Examples of mismatch repair proteins and nucleic acid sequences include the following: PMS2 (mouse) (SEQ ID NO:5) MEQTEGVSTE CAKAIKPIDG KSVHQICSGQ VILSLSTAVK ELIENSVDAG ATTIDLRLKD   60 YGVDLIEVSD NGCGVEEENF EGLALKHHTS KIQEFADLTQ VETFGFRGEA LSSLCALSDV  120 TISTCHGSAS VGTRLVFDHN GKITQKTPYP RPKGTTVSVQ HLFYTLPVRY KEFQRNIKKE  180 YSKMVQVLQA YCIISAGVRV SCTNQLGQGK RHAVVCTSGT SGMKENIGSV FGQKQLQSLI  240 PFVQLPPSDA VCEEYGLSTS GRHKTFSTFR ASFHSARTAP GGVQQTGSFS SSIRGPVTQQ  300 RSLSLSMRFY HMYNRHQYPF VVLNVSVDSE CVDINVTPDK RQILLQEEKL LLAVLKTSLI  360 GMFDSDANKL NVNQQPLLDV EGNLVKLHTA ELEKPVPGKQ DNSPSLKSTA DEKRVASISR  420 LREAFSLHPT KEIKSRGPET AELTRSFPSE KRGVLSSYPS DVISYRGLRG SQDKLVSPTD  480 SPGDCMDREK IEKDSGLSST SAGSEEEFST PEVASSFSSD YNVSSLEDRP SQETINCGDL  540 DCRPPGTGQS LKPEDHGYQC KALPLARLSP TNAKRFKTEE RPSNVNISQR LPGPQSTSAA  600 EVDVAIKMNK RIVLLEFSLS SLAKRMKQLQ HLKAQNKHEL SYRKFRAKIC PGENQAAEDE  660 LRKEISKSMF AEMEILGQFN LGFIVTKLKE DLFLVDQHAA DEKYNFEMLQ QHTVLQAQRL  720 ITPQTLNLTA VNEAVLIENL EIFRKNGFDF VIDEDAPVTE RAKLISLPTS KNWTFGPQDI  780 DELIFMLSDS PGVMCRPSRV RQMFASRACR KSVMIGTALN ASEMKKLITH MGEMDHPWNC  840 PHGRPTMRHV ANLDVISQN  859 PMS2 (mouse cDNA) (SEQ ID NO:6) gaattccggt gaaggtcctg aagaatttcc agattcctga gtatcattgg aggagacaga   60 taacctgtcg tcaggtaacg atggtgtata tgcaacagaa atgggtgttc ctggagacgc  120 gtcttttccc gagagcggca ccgcaactct cccgcggtga ctgtgactgg aggagtcctg  180 catccatgga gcaaaccgaa ggcgtgagta cagaatgtgc taaggccatc aagcctattg  240 atgggaagtc agtccatcaa atttgttctg ggcaggtgat actcagttta agcaccgctg  300 tgaaggagtt gatagaaaat agtgtagatg ctggtgctac tactattgat ctaaggctta  360 aagactatgg ggtggacctc attgaagttt cagacaatgg atgtggggta gaagaagaaa  420 actttgaagg tctagctctg aaacatcaca catctaagat tcaagagttt gccgacctca  480 cgcaggttga aactttcggc tttcgggggg aagctctgag ctctctgtgt gcactaagtg  540 atgtcactat atctacctgc cacgggtctg caagcgttgg gactcgactg gtgtttgacc  600 ataatgggaa aatcacccag aaaactccct acccccgacc taaaggaacc acagtcagtg  660 tgcagcactt attttataca ctacccgtgc gttacaaaga gtttcagagg aacattaaaa  720 aggagtattc caaaatggtg caggtcttac aggcgtactg tatcatctca gcaggcgtcc  780 gtgtaagctg cactaatcag ctcggacagg ggaagcggca cgctgtggtg tgcacaagcg  840 gcacgtctgg catgaaggaa aatatcgggt ctgtgtttgg ccagaagcag ttgcaaagcc  900 tcattccttt tgttcagctg ccccctagtg acgctgtgtg tgaagagtac ggcctgagca  960 cttcaggacg ccacaaaacc ttttctacgt ttcgggcttc atttcacagt gcacgcacgg 1020 cgccgggagg agtgcaacag acaggcagtt tttcttcatc aatcagaggc cctgtgaccc 1080 agcaaaggtc tctaagcttg tcaatgaggt tttatcacat gtataaccgg catcagtacc 1140 catttgtcgt ccttaacgtt tccgttgact cagaatgtgt ggatattaat gtaactccag 1200 ataaaaggca aattctacta caagaagaga agctattgct ggccgtttta aagacctcct 1260 tgataggaat gtttgacagt gatgcaaaca agcttaatgt caaccagcag ccactgctag 1320 atgttgaagg taacttagta aagctgcata ctgcagaact agaaaagcct gtgccaggaa 1380 agcaagataa ctctccttca ctgaagagca cagcagacga gaaaagggta gcatccatct 1440 ccaggctgag agaggccttt tctcttcatc ctactaaaga gatcaagtct aggggtccag 1500 agactgctga actgacacgg agttttccaa gtgagaaaag gggcgtgtta tcctcttatc 1560 cttcagacgt catctcttac agaggcctcc gtggctcgca ggacaaattg gtgagtccca 1620 cggacagccc tggtgactgt atggacagag agaaaataga aaaagactca gggctcagca 1680 gcacctcagc tggctctgag gaagagttca gcaccccaga agtggccagt agctttagca 1740 gtgactataa cgtgagctcc ctagaagaca gaccttctca ggaaaccata aactgtggtg 1800 acctggactg ccgtcctcca ggtacaggac agtccttgaa gccagaagac catggatatc 1860 aatgcaaagc tctacctcta gctcgtctgt cacccacaaa tgccaagcgc ttcaagacag 1920 aggaaagacc ctcaaatgtc aacatttctc aaagattgcc tggtcctcag agcacctcag 1980 cagctgaggt cgatgtagcc ataaaaatga ataagagaat cgtgctcctc gagttctctc 2040 tgagttctct agctaagcga atgaagcagt tacagcacct aaaggcgcag aacaaacatg 2100 aactgagtta cagaaaattt agggccaaga tttgccctgg agaaaaccaa gcagcagaag 2160 atgaactcag aaaagagatt agtaaatcga tgtttgcaga gatggagatc ttgggtcagt 2220 ttaacctggg atttatagta accaaactga aagaggacct cttcctggtg gaccagcatg 2280 ctgcggatga gaagtacaac tttgagatgc tgcagcagca cacggtgctc caggcgcaga 2340 ggctcatcac accccagact ctgaacttaa ctgctgtcaa tgaagctgta ctgatagaaa 2400 atctggaaat attcagaaag aatggctttg actttgtcat tgatgaggat gctccagtca 2460 ctgaaagggc taaattgatt tccttaccaa ctagtaaaaa ctggaccttt ggaccccaag 2520 atatagatga actgatcttt atgttaagtg acagccctgg ggtcatgtgc cggccctcac 2580 gagtcagaca gatgtttgct tccagagcct gtcggaagtc agtgatgatt ggaacggcgc 2640 tcaatgcgag cgagatgaag aagctcatca cccacatggg tgagatggac cacccctgga 2700 actgccccca cggcaggcca accatgaggc acgttgccaa tctggatgtc atctctcaga 2760 actgacacac cccttgtagc atagagttta ttacagattg ttcggtttgc aaagagaagg 2820 ttttaagtaa tctgattatc gttgtacaaa aattagcatg ctgctttaat gtactggatc 2880 catttaaaag cagtgttaag gcaggcatga tggagtgttc ctctagctca gctacttggg 2940 tgatccggtg ggagctcatg tgagcccagg actttgagac cactccgagc cacattcatg 3000 agactcaatt caaggacaaa aaaaaaaaga tatttttgaa gccttttaaa aaaaaa 3056 PMS2 (human) (SEQ ID NO:7) MERAESSSTE PAKAIKPIDR KSVHQICSGQ VVLSLSTAVK ELVENSLDAG ATNIDLKLKD   60 YGVDLIEVSD NGCGVEEENF EGLTLKHHTS KIQEFADLTQ VETFGFRGEA LSSLCALSDV  120 TISTCHASAK VGTRLMFDHN GKIIQKTPYP RPRGTTVSVQ QLFSTLPVRH KEFQRNIKKE  180 YAKMVQVLHA YCIISAGIRV SCTNQLGQGK RQPVVCTGGS PSIKENIGSV FGQKQLQSLI  240 PFVQLPPSDS VCEEYGLSCS DALHNLFYIS GFISQCTHGV GRSSTDRQFF FINRRPCDPA  300 KVCRLVNEVY HMYNRHQYPF VVLNISVDSE CVDINVTPDK RQILLQEEKL LLAVLKTSLI  360 GMFDSDVNKL NVSQQPLLDV EGNLIKMHAA DLEKPMVEKQ DQSPSLRTGE EKKDVSISRL  420 REAFSLRHTT ENKPHSPKTP EPRRSPLGQK RGMLSSSTSG AISDKGVLRP QKEAVSSSHG  480 PSDPTDRAEV EKDSGHGSTS VDSEGFSIPD TGSHCSSEYA ASSPGDRGSQ EHVDSQEKAP  540 ETDDSFSDVD CHSNQEDTGC KFRVLPQPTN LATPNTKRFK KEEILSSSDI CQKLVNTQDM  600 SASQVDVAVK INKKVVPLDF SMSSLAKRIK QLHHEAQQSE GEQNYRKFPA KICPGENQAA  660 EDELRKEISK TMFAEMEIIG QFNLGFIITK LNEDIFIVDQ HATDEKYNFE MLQQHTVLQG  720 QRLIAPQTLN LTAVNEAVLI ENLEIFRKNG FDFVIDENAP VTERAKLISL PTSKNWTFGP  780 QDVDELIFML SDSPGVMCRP SRVKQMFASR ACRKSVMIGI ALNTSEMKKL ITHMGEMDHP  840 WNCPHGRPTM RHIANLGVIS QN  862 PMS2 (human cDNA) (SEQ ID NO:8) cgaggcggat cgggtgttgc atccatggag cgagctgaga gctcgagtac agaacctgct   60 aaggccatca aacctattga tcggaagtca gtccatcaga tttgctctgg gcaggtggta  120 ctgagtctaa gcactgcggt aaaggagtta gtagaaaaca gtctggatgc tggtgccact  180 aatattgatc taaagcttaa ggactatgga gtggatctta ttgaagtttc agacaatgga  240 tgtggggtag aagaagaaaa cttcgaaggc ttaactctga aacatcacac atctaagatt  300 caagagtttg ccgacctaac tcaggttgaa acttttggct ttcgggggga agctctgagc  360 tcactttgtg cactgagcga tgtcaccatt tctacctgcc acgcatcggc gaaggttgga  420 actcgactga tgtttgatca caatgggaaa attatccaga aaacccccta cccccgcccc  480 agagggacca cagtcagcgt gcagcagtta ttttccacac tacctgtgcg ccataaggaa  540 tttcaaagga atattaagaa ggagtatgcc aaaatggtcc aggtcttaca tgcatactgt  600 atcatttcag caggcatccg tgtaagttgc accaatcagc ttggacaagg aaaacgacag  660 cctgtggtat gcacaggtgg aagccccagc ataaaggaaa atatcggctc tgtgtttggg  720 cagaagcagt tgcaaagcct cattcctttt gttcagctgc cccctagtga ctccgtgtgt  780 gaagagtacg gtttgagctg ttcggatgct ctgcataatc ttttttacat ctcaggtttc  840 atttcacaat gcacgcatgg agttggaagg agttcaacag acagacagtt tttctttatc  900 aaccggcggc cttgtgaccc agcaaaggtc tgcagactcg tgaatgaggt ctaccacatg  960 tataatcgac accagtatcc atttgttgtt cttaacattt ctgttgattc agaatgcgtt 1020 gatatcaatg ttactccaga taaaaggcaa attttgctac aagaggaaaa gcttttgttg 1080 gcagttttaa agacctcttt gataggaatg tttgatagtg atgtcaacaa gctaaatgtc 1140 agtcagcagc cactgctgga tgttgaaggt aacttaataa aaatgcatgc agcggatttg 1200 gaaaagccca tggtagaaaa gcaggatcaa tccccttcat taaggactgg agaagaaaaa 1260 aaagacgtgt ccatttccag actgcgagag gccttttctc ttcgtcacac aacagagaac 1320 aagcctcaca gcccaaagac tccagaacca agaaggagcc ctctaggaca gaaaaggggt 1380 atgctgtctt ctagcacttc aggtgccatc tctgacaaag gcgtcctgag acctcagaaa 1440 gaggcagtga gttccagtca cggacccagt gaccctacgg acagagcgga ggtggagaag 1500 gactcggggc acggcagcac ttccgtggat tctgaggggt tcagcatccc agacacgggc 1560 agtcactgca gcagcgagta tgcggccagc tccccagggg acaggggctc gcaggaacat 1620 gtggactctc aggagaaagc gcctgaaact gacgactctt tttcagatgt ggactgccat 1680 tcaaaccagg aagataccgg atgtaaattt cgagttttgc ctcagccaac taatctcgca 1740 accccaaaca caaagcgttt taaaaaagaa gaaattcttt ccagttctga catttgtcaa 1800 aagttagtaa atactcagga catgtcagcc tctcaggttg atgtagctgt gaaaattaat 1860 aagaaagttg tgcccctgga cttttctatg agttctttag ctaaacgaat aaagcagtta 1920 catcatgaag cacagcaaag tgaaggggaa cagaattaca ggaagtttag ggcaaagatt 1980 tgtcctggag aaaatcaagc agccgaagat gaactaagaa aagagataag taaaacgatg 2040 tttgcagaaa tggaaatcat tggtcagttt aacctgggat ttataataac caaactgaat 2100 gaggatatct tcatagtgga ccagcatgcc acggacgaga agtataactt cgagatgctg 2160 cagcagcaca ccgtgctcca ggggcagagg ctcatagcac ctcagactct caacttaact 2220 gctgttaatg aagctgttct gatagaaaat ctggaaatat ttagaaagaa tggctttgat 2280 tttgttatcg atgaaaatgc tccagtcact gaaagggcta aactgatttc cttgccaact 2340 agtaaaaact ggaccttcgg accccaggac gtcgatgaac tgatcttcat gctgagcgac 2400 agccctgggg tcatgtgccg gccttcccga gtcaagcaga tgtttgcctc cagagcctgc 2460 cggaagtcgg tgatgattgg gactgctctt aacacaagcg agatgaagaa actgatcacc 2520 cacatggggg agatggacca cccctggaac tgtccccatg gaaggccaac catgagacac 2580 atcgccaacc tgggtgtcat ttctcagaac tgaccgtagt cactgtatgg aataattggt 2640 tttatcgcag atttttatgt tttgaaagac agagtcttca ctaacctttt ttgttttaaa 2700 atgaaacctg ctacttaaaa aaaatacaca tcacacccat ttaaaagtga tcttgagaac 2760 cttttcaaac c 2771 PMS1 (human) (SEQ ID NO:9) MKQLPAATVR LLSSSQIITS VVSVVKELIE NSLDAGATSV DVKLENYGFD KIEVRDNGEG   60 IKAVDAPVMA MKYYTSKINS HEDLENLTTY GFRGEALGSI CCIAEVLITT RTAADNFSTQ  120 YVLDGSGHIL SQKPSHLGQG TTVTALRLFK NLPVRKQFYS TAKKCKDEIK KIQDLLMSFG  180 ILKPDLRIVF VHNKAVTWQK SRVSDHKMAL MSVLGTAVMN NMESFQYHSE ESQIYLSGFL  240 PKCDADHSFT SLSTPERSFI FINSRPVHQK DILKLIRHHY NLKCLKESTR LYPVFFLKID  300 VPTADVDVNL TPDKSQVLLQ NKESVLIALE NLMTTCYGPL PSTNSYENNK TDVSAADIVL  360 SKTAETDVLF NKVESSGKNY SNVDTSVIPF QNDMHNDESG KNTDDCLNHQ ISIGDFGYGH  420 CSSEISNIDK NTKNAFQDIS MSNVSWENSQ TEYSKTCFIS SVKHTQSENG NKDHIDESGE  480 NEEEAGLENS SEISADEWSR GNILKNSVGE NIEPVKILVP EKSLPCKVSN NNYPIPEQMN  540 LNEDSCNKKS NVIDNKSGKV TAYDLLSNRV IKKPMSASAL FVQDHRPQFL IENPKTSLED  600 ATLQIEELWK TLSEEEKLKY EEKATKDLER YNSQMKRAIE QESQMSLKDG RKKIKPTSAW  660 NLAQKHKLKT SLSNQPKLDE LLQSQIEKRR SQNIKMVQIP FSMKNLKINF KKQNKVDLEE  720 KDEPCLIHNL RFPDAWLMTS KTEVMLLNPY RVEEALLFKR LLENHKLPAE PLEKPIMLTE  780 SLFNGSHYLD VLYKMTADDQ RYSGSTYLSD PRLTANGFKI KLIPGVSITE NYLEIEGMAN  840 CLPFYGVADL KEILNAILNR NAKEVYECRP RKVISYLEGE AVRLSRQLPM YLSKEDIQDI  900 IYRMKHQFGN EIKECVHGRP FFHHLTYLPE TT  932 PMS1 (human) (SEQ ID NO:10) ggcacgagtg gctgcttgcg gctagtggat ggtaattgcc tgcctcgcgc tagcagcaag   60 ctgctctgtt aaaagcgaaa atgaaacaat tgcctgcggc aacagttcga ctcctttcaa  120 gttctcagat catcacttcg gtggtcagtg ttgtaaaaga gcttattgaa aactccttgg  180 atgctggtgc cacaagcgta gatgttaaac tggagaacta tggatttgat aaaattgagg  240 tgcgagataa cggggagggt atcaaggctg ttgatgcacc tgtaatggca atgaagtact  300 acacctcaaa aataaatagt catgaagatc ttgaaaattt gacaacttac ggttttcgtg  360 gagaagcctt ggggtcaatt tgttgtatag ctgaggtttt aattacaaca agaacggctg  420 ctgataattt tagcacccag tatgttttag atggcagtgg ccacatactt tctcagaaac  480 cttcacatct tggtcaaggt acaactgtaa ctgctttaag attatttaag aatctacctg  540 taagaaagca gttttactca actgcaaaaa aatgtaaaga tgaaataaaa aagatccaag  600 atctcctcat gagctttggt atccttaaac ctgacttaag gattgtcttt gtacataaca  660 aggcagttat ttggcagaaa agcagagtat cagatcacaa gatggctctc atgtcagttc  720 tggggactgc tgttatgaac aatatggaat cctttcagta ccactctgaa gaatctcaga  780 tttatctcag tggatttctt ccaaagtgtg atgcagacca ctctttcact agtctttcaa  840 caccagaaag aagtttcatc ttcataaaca gtcgaccagt acatcaaaaa gatatcttaa  900 agttaatccg acatcattac aatctgaaat gcctaaagga atctactcgt ttgtatcctg  960 ttttctttct gaaaatcgat gttcctacag ctgatgttga tgtaaattta acaccagata 1020 aaagccaagt attattacaa aataaggaat ctgttttaat tgctcttgaa aatctgatga 1080 cgacttgtta tggaccatta cctagtacaa attcttatga aaataataaa acagatgttt 1140 ccgcagctga catcgttctt agtaaaacag cagaaacaga tgtgcttttt aataaagtgg 1200 aatcatctgg aaagaattat tcaaatgttg atacttcagt cattccattc caaaatgata 1260 tgcataatga tgaatctgga aaaaacactg atgattgttt aaatcaccag ataagtattg 1320 gtgactttgg ttatggtcat tgtagtagtg aaatttctaa cattgataaa aacactaaga 1380 atgcatttca ggacatttca atgagtaatg tatcatggga gaactctcag acggaatata 1440 gtaaaacttg ttttataagt tccgttaagc acacccagtc agaaaatggc aataaagacc 1500 atatagatga gagtggggaa aatgaggaag aagcaggtct tgaaaactct tcggaaattt 1560 ctgcagatga gtggagcagg ggaaatatac ttaaaaattc agtgggagag aatattgaac 1620 ctgtgaaaat tttagtgcct gaaaaaagtt taccatgtaa agtaagtaat aataattatc 1680 caatccctga acaaatgaat cttaatgaag attcatgtaa caaaaaatca aatgtaatag 1740 ataataaatc tggaaaagtt acagcttatg atttacttag caatcgagta atcaagaaac 1800 ccatgtcagc aagtgctctt tttgttcaag atcatcgtcc tcagtttctc atagaaaatc 1860 ctaagactag tttagaggat gcaacactac aaattgaaga actgtggaag acattgagtg 1920 aagaggaaaa actgaaatat gaagagaagg ctactaaaga cttggaacga tacaatagtc 1980 aaatgaagag agccattgaa caggagtcac aaatgtcact aaaagatggc agaaaaaaga 2040 taaaacccac cagcgcatgg aatttggccc agaagcacaa gttaaaaacc tcattatcta 2100 atcaaccaaa acttgatgaa ctccttcagt cccaaattga aaaaagaagg agtcaaaata 2160 ttaaaatggt acagatcccc ttttctatga aaaacttaaa aataaatttt aagaaacaaa 2220 acaaagttga cttagaagag aaggatgaac cttgcttgat ccacaatctc aggtttcctg 2280 atgcatggct aatgacatcc aaaacagagg taatgttatt aaatccatat agagtagaag 2340 aagccctgct atttaaaaga cttcttgaga atcataaact tcctgcagag ccactggaaa 2400 agccaattat gttaacagag agtcttttta atggatctca ttatttagac gttttatata 2460 aaatgacagc agatgaccaa agatacagtg gatcaactta cctgtctgat cctcgtctta 2520 cagcgaatgg tttcaagata aaattgatac caggagtttc aattactgaa aattacttgg 2580 aaatagaagg aatggctaat tgtctcccat tctatggagt agcagattta aaagaaattc 2640 ttaatgctat attaaacaga aatgcaaagg aagtttatga atgtagacct cgcaaagtga 2700 taagttattt agagggagaa gcagtgcgtc tatccagaca attacccatg tacttatcaa 2760 aagaggacat ccaagacatt atctacagaa tgaagcacca gtttggaaat gaaattaaag 2820 agtgtgttca tggtcgccca ttttttcatc atttaaccta tcttccagaa actacatgat 2880 taaatatgtt taagaagatt agttaccatt gaaattggtt ctgtcataaa acagcatgag 2940 tctggtttta aattatcttt gtattatgtg tcacatggtt attttttaaa tgaggattca 3000 ctgacttgtt tttatattga aaaaagttcc acgtattgta gaaaacgtaa ataaactaat 3060 aac 3063 MSH2 (human) (SEQ ID NO:11) MAVQPKETLQ LESAAEVGFV RFFQGMPEKP TTTVRLFDRG DFYTAHGEDA LLAAREVFKT   60 QGVIKYMGPA GAKNLQSVVL SKMNFESFVK DLLLVRQYRV EVYKNRAGNK ASKENDWYLA  120 YKASPGNLSQ FEDILFGNND MSASIGVVGV KMSAVDGQRQ VGVGYVDSIQ RKLGLCEFPD  180 NDQFSNLEAL LIQIGPKECV LPGGETAGDM GKLRQIIQRG GILITERKKA DFSTKDIYQD  240 LNRLLKGKKG EQMNSAVLPE MENQVAVSSL SAVIKFLELL SDDSNFGQFE LTTFDFSQYM  300 KLDIAAVRAL NLFQGSVEDT TGSQSLAALL NKCKTPQGQR LVNQWIKQPL MDKNRIEERL  360 NLVEAFVEDA ELRQTLQEDL LRRFPDLNRL AKKFQRQAAN LQDCYRLYQG INQLPNVIQA  420 LEKHEGKHQK LLLAVFVTPL TDLRSDFSKF QEMIETTLDM DQVENHEFLV KPSFDPNLSE  480 LREIMNDLEK KMQSTLISAA RDLGLDPGKQ IKLDSSAQFG YYFRVTCKEE KVLRNNKNFS  540 TVDIQKNGVK FTNSKLTSLN EEYTKNKTEY EEAQDAIVKE IVNISSGYVE PMQTLNDVLA  600 QLDAVVSFAH VSNGAPVPYV RPAILEKGQG RIILKASRHA CVEVQDEIAF IPNDVYFEKD  660 KQMFHIITGP NMGGKSTYIR QTGVIVLMAQ IGCFVPCESA EVSIVDCILA RVGAGDSQLK  720 GVSTFMAEML ETASILRSAT KDSLIIIDEL GRGTSTYDGF GLAWAISEYI ATKIGAFCMF  780 ATHFHELTAL ANQIPTVNNL HVTALTTEET LTMLYQVKKG VCDQSFGIHV AELANFPKHV  840 IECAKQKALE LEEFQYIGES QGYDIMEPAA KKCYLEREQG EKIIQEFLSK VKQMPFTEMS  900 EENITIKLKQ LKAEVIAKNN SFVNEIISRI KVTT  934 MSH2 (human cDNA) (SEQ ID NO:12) ggcgggaaac agcttagtgg gtgtggggtc gcgcattttc ttcaaccagg aggtgaggag   60 gtttcgacat ggcggtgcag ccgaaggaga cgctgcagtt ggagagcgcg gccgaggtcg  120 gcttcgtgcg cttctttcag ggcatgccgg agaagccgac caccacagtg cgccttttcg  180 accggggcga cttctatacg gcgcacggcg aggacgcgct gctggccgcc cgggaggtgt  240 tcaagaccca gggggtgatc aagtacatgg ggccggcagg agcaaagaat ctgcagagtg  300 ttgtgcttag taaaatgaat tttgaatctt ttgtaaaaga tcttcttctg gttcgtcagt  360 atagagttga agtttataag aatagagctg gaaataaggc atccaaggag aatgattggt  420 atttggcata taaggcttct cctggcaatc tctctcagtt tgaagacatt ctctttggta  480 acaatgatat gtcagcttcc attggtgttg tgggtgttaa aatgtccgca gttgatggcc  540 agagacaggt tggagttggg tatgtggatt ccatacagag gaaactagga ctgtgtgaat  600 tccctgataa tgatcagttc tccaatcttg aggctctcct catccagatt ggaccaaagg  660 aatgtgtttt acccggagga gagactgctg gagacatggg gaaactgaga cagataattc  720 aaagaggagg aattctgatc acagaaagaa aaaaagctga cttttccaca aaagacattt  780 atcaggacct caaccggttg ttgaaaggca aaaagggaga gcagatgaat agtgctgtat  840 tgccagaaat ggagaatcag gttgcagttt catcactgtc tgcggtaatc aagtttttag  900 aactcttatc agatgattcc aactttggac agtttgaact gactactttt gacttcagcc  960 agtatatgaa attggatatt gcagcagtca gagcccttaa cctttttcag ggttctgttg 1020 aagataccac tggctctcag tctctggctg ccttgctgaa taagtgtaaa acccctcaag 1080 gacaaagact tgttaaccag tggattaagc agcctctcat ggataagaac agaatagagg 1140 agagattgaa tttagtggaa gcttttgtag aagatgcaga attgaggcag actttacaag 1200 aagatttact tcgtcgattc ccagatctta accgacttgc caagaagttt caaagacaag 1260 cagcaaactt acaagattgt taccgactct atcagggtat aaatcaacta cctaatgtta 1320 tacaggctct ggaaaaacat gaaggaaaac accagaaatt attgttggca gtttttgtga 1380 ctcctcttac tgatcttcgt tctgacttct ccaagtttca ggaaatgata gaaacaactt 1440 tagatatgga tcaggtggaa aaccatgaat tccttgtaaa accttcattt gatcctaatc 1500 tcagtgaatt aagagaaata atgaatgact tggaaaagaa gatgcagtca acattaataa 1560 gtgcagccag agatcttggc ttggaccctg gcaaacagat taaactggat tccagtgcac 1620 agtttggata ttactttcgt gtaacctgta aggaagaaaa agtccttcgt aacaataaaa 1680 actttagtac tgtagatatc cagaagaatg gtgttaaatt taccaacagc aaattgactt 1740 ctttaaatga agagtatacc aaaaataaaa cagaatatga agaagcccag gatgccattg 1800 ttaaagaaat tgtcaatatt tcttcaggct atgtagaacc aatgcagaca ctcaatgatg 1860 tgttagctca gctagatgct gttgtcagct ttgctcacgt gtcaaatgga gcacctgttc 1920 catatgtacg accagccatt ttggagaaag gacaaggaag aattatatta aaagcatcca 1980 ggcatgcttg tgttgaagtt caagatgaaa ttgcatttat tcctaatgac gtatactttg 2040 aaaaagataa acagatgttc cacatcatta ctggccccaa tatgggaggt aaatcaacat 2100 atattcgaca aactggggtg atagtactca tggcccaaat tgggtgtttt gtgccatgtg 2160 agtcagcaga agtgtccatt gtggactgca tcttagcccg agtaggggct ggtgacagtc 2220 aattgaaagg agtctccacg ttcatggctg aaatgttgga aactgcttct atcctcaggt 2280 ctgcaaccaa agattcatta ataatcatag atgaattggg aagaggaact tctacctacg 2340 atggatttgg gttagcatgg gctatatcag aatacattgc aacaaagatt ggtgcttttt 2400 gcatgtttgc aacccatttt catgaactta ctgccttggc caatcagata ccaactgtta 2460 ataatctaca tgtcacagca ctcaccactg aagagacctt aactatgctt tatcaggtga 2520 agaaaggtgt ctgtgatcaa agttttggga ttcatgttgc agagcttgct aatttcccta 2580 agcatgtaat agagtgtgct aaacagaaag ccctggaact tgaggagttt cagtatattg 2640 gagaatcgca aggatatgat atcatggaac cagcagcaaa gaagtgctat ctggaaagag 2700 agcaaggtga aaaaattatt caggagttcc tgtccaaggt gaaacaaatg ccctttactg 2760 aaatgtcaga agaaaacatc acaataaagt taaaacagct aaaagctgaa gtaatagcaa 2820 agaataatag ctttgtaaat gaaatcattt cacgaataaa agttactacg tgaaaaatcc 2880 cagtaatgga atgaaggtaa tattgataag ctattgtctg taatagtttt atattgtttt 2940 atattaaccc tttttccata gtgttaactg tcagtgccca tgggctatca acttaataag 3000 atatttagta atattttact ttgaggacat tttcaaagat ttttattttg aaaaatgaga 3060 gctgtaactg aggactgttt gcaattgaca taggcaataa taagtgatgt gctgaatttt 3120 ataaataaaa tcatgtagtt tgtgg 3145 MLH1 (human) (SEQ ID NO:13) MSFVAGVIRR LDETVVNRIA AGEVIQRPAN AIKEMIENCL DAKSTSIQVI VKEGGLKLIQ   60 IQDNGTGIRK EDLDIVCERF TTSKLQSFED LASISTYGFR GEALASISHV AHVTITTKTA  120 DGKCAYRASY SDGKLKAPPK PCAGNQGTQI TVEDLFYNIA TRRKALKNPS EEYGKILEVV  180 GRYSVHNAGI SFSVKKQGET VADVRTLPNA STVDNIRSIF GNAVSRELIE IGCEDKTLAF  240 KMNGYISNAN YSVKKCIFLL FINHRLVEST SLRKAIETVY AAYLPKNTHP FLYLSLEISP  300 QNVDVNVHPT KHEVHFLHEE SILERVQQHI ESKLLGSNSS RMYFTQTLLP GLAGPSGEMV  360 KSTTSLTSSS TSGSSDKVYA HQMVRTDSRE QKLDAFLQPL SKPLSSQPQA IVTEDKTDIS  420 SGRARQQDEE MLELPAPAEV AAKNQSLEGD TTKGTSEMSE KRGPTSSNPR KRHREDSDVE  480 MVEDDSRKEM TAACTPRRRI INLTSVLSLQ EEINEQGHEV LREMLHNHSF VGCVNPQWAL  540 AQHQTKLYLL NTTKLSEELF YQILIYDFAN FGVLRLSEPA PLFDLAMLAL DSPESGWTEE  600 DGPKEGLAEY IVEFLKKKAE MLADYFSLEI DEEGNLIGLP LLIDNYVPPL EGLPIFILRL  660 ATEVNWDEEK ECFESLSKEC AMFYSIRKQY ISEESTLSGQ QSEVPGSIPN SWKWTVEHIV  720 YKALRSHILP PKHFTEDGNI LQLANLPDLY KVFERC  756 MLH1 (human) (SEQ ID NO:14) cttggctctt ctggcgccaa aatgtcgttc gtggcagggg ttattcggcg gctggacgag   60 acagtggtga accgcatcgc ggcgggggaa gttatccagc ggccagctaa tgctatcaaa  120 gagatgattg agaactgttt agatgcaaaa tccacaagta ttcaagtgat tgttaaagag  180 ggaggcctga agttgattca gatccaagac aatggcaccg ggatcaggaa agaagatctg  240 gatattgtat gtgaaaggtt cactactagt aaactgcagt cctttgagga tttagccagt  300 atttctacct atggctttcg aggtgaggct ttggccagca taagccatgt ggctcatgtt  360 actattacaa cgaaaacagc tgatggaaag tgtgcataca gagcaagtta ctcagatgga  420 aaactgaaag cccctcctaa accatgtgct ggcaatcaag ggacccagat cacggtggag  480 gacctttttt acaacatagc cacgaggaga aaagctttaa aaaatccaag tgaagaatat  540 gggaaaattt tggaagttgt tggcaggtat tcagtacaca atgcaggcat tagtttctca  600 gttaaaaaac aaggagagac agtagctgat gttaggacac tacccaatgc ctcaaccgtg  660 gacaatattc gctccatctt tggaaatgct gttagtcgag aactgataga aattggatgt  720 gaggataaaa ccctagcctt caaaatgaat ggttacatat ccaatgcaaa ctactcagtg  780 aagaagtgca tcttcttact cttcatcaac catcgtctgg tagaatcaac ttccttgaga  840 aaagccatag aaacagtgta tgcagcctat ttgcccaaaa acacacaccc attcctgtac  900 ctcagtttag aaatcagtcc ccagaatgtg gatgttaatg tgcaccccac aaagcatgaa  960 gttcacttcc tgcacgagga gagcatcctg gagcgggtgc agcagcacat cgagagcaag 1020 ctcctgggct ccaattcctc caggatgtac ttcacccaga ctttgctacc aggacttgct 1080 ggcccctctg gggagatggt taaatccaca acaagtctga cctcgtcttc tacttctgga 1140 agtagtgata aggtctatgc ccaccagatg gttcgtacag attcccggga acagaagctt 1200 gatgcatttc tgcagcctct gagcaaaccc ctgtccagtc agccccaggc cattgtcaca 1260 gaggataaga cagatatttc tagtggcagg gctaggcagc aagatgagga gatgcttgaa 1320 ctcccagccc ctgctgaagt ggctgccaaa aatcagagct tggaggggga tacaacaaag 1380 gggacttcag aaatgtcaga gaagagagga cctacttcca gcaaccccag aaagagacat 1440 cgggaagatt ctgatgtgga aatggtggaa gatgattccc gaaaggaaat gactgcagct 1500 tgtacccccc ggagaaggat cattaacctc actagtgttt tgagtctcca ggaagaaatt 1560 aatgagcagg gacatgaggt tctccgggag atgttgcata accactcctt cgtgggctgt 1620 gtgaatcctc agtgggcctt ggcacagcat caaaccaagt tataccttct caacaccacc 1680 aagcttagtg aagaactgtt ctaccagata ctcatttatg attttgccaa ttttggtgtt 1740 ctcaggttat cggagccagc accgctcttt gaccttgcca tgcttgcctt agatagtcca 1800 gagagtggct ggacagagga agatggtccc aaagaaggac ttgctgaata cattgttgag 1860 tttctgaaga agaaggctga gatgcttgca gactatttct ctttggaaat tgatgaggaa 1920 gggaacctga ttggattacc ccttctgatt gacaactatg tgcccccttt ggagggactg 1980 cctatcttca ttcttcgact agccactgag gtgaattggg acgaagaaaa ggaatgtttt 2040 gaaagcctca gtaaagaatg cgctatgttc tattccatcc ggaagcagta catatctgag 2100 gagtcgaccc tctcaggcca gcagagtgaa gtgcctggct ccattccaaa ctcctggaag 2160 tggactgtgg aacacattgt ctataaagcc ttgcgctcac acattctgcc tcctaaacat 2220 ttcacagaag atggaaatat cctgcagctt gctaacctgc ctgatctata caaagtcttt 2280 gagaggtgtt aaatatggtt atttatgcac tgtgggatgt gttcttcttt ctctgtattc 2340 cgatacaaag tgttgtatca aagtgtgata tacaaagtgt accaacataa gtgttggtag 2400 cacttaagac ttatacttgc cttctgatag tattccttta tacacagtgg attgattata 2460 aataaataga tgtgtcttaa cata 2484 hPMS2-134 (human) (SEQ ID NO:15) MERAESSSTE PAKAIKPIDR KSVHQICSGQ VVLSLSTAVK ELVENSLDAG ATNIDLKLKD   60 YGVDLIEVSD NGCGVEEENF EGLTLKHHTS KIQEFADLTQ VETFGFRGEA LSSLCALSDV  120 TISTCHASAK VGT  133 hPMS2-134 (human cDNA) (SEQ ID NO:16) cgaggcggat cgggtgttgc atccatggag cgagctgaga gctcgagtac agaacctgct   60 aaggccatca aacctattga tcggaagtca gtccatcaga tttgctctgg gcaggtggta  120 ctgagtctaa gcactgcggt aaaggagtta gtagaaaaca gtctggatgc tggtgccact  180 aatattgatc taaagcttaa ggactatgga gtggatctta ttgaagtttc agacaatgga  240 tgtggggtag aagaagaaaa cttcgaaggc ttaactctga aacatcacac atctaagatt  300 caagagtttg ccgacctaac tcaggttgaa acttttggct ttcgggggga agctctgagc  360 tcactttgtg cactgagcga tgtcaccatt tctacctgcc acgcatcggc gaaggttgga  420 acttga  426

[0056] For further information on the background of the invention the following references may be consulted, each of which is incorporated herein by reference in its entirety:

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[0097] The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.

EXAMPLE 1 Stable Expression of Dominant Negative MMR Genes in Cells Results in Widespread Mutations of a Reporter Gene and its Encoded Polypeptide

[0098] Expression of a dominant negative allele in an otherwise MMR proficient cell could render these host cells MMR deficient (Nicolaides, N. C. et al. (1998) Mol. Cell. Biol. 18:1635-1641). The creation of MMR deficient cells can lead to the generation of genetic alterations throughout the entire genome of a host organisms offspring, yielding a population of genetically altered offspring or siblings that may produce biochemicals with altered properties. This patent application teaches of the use of dominant negative MMR genes in antigen-producing cells, including but not limited to rodent, human, primate, yeast, insect, and prokaryotic cells producing proteins that may serve as therapeutic antigens for vaccination. The cell expression systems described above that are used to produce antigens are well known by those skilled in the art of vaccine therapeutics.

[0099] To demonstrate the ability to create MMR defective mammalian cells using dominant negative alleles of MMR genes, we first transfected a MMR proficient rodent cell line with an expression vector containing the human the previously published dominant negative PMS2 mutant referred herein as PMS134 (cell line referred to as TKPMS134), or with no insert (cell line referred to as TKvec). A fragment containing the PMS134 cDNA was cloned into the pEF expression vector which contains the constitutively active elongation factor promoter along with the neomycin resistance gene as selectable marker. The results showed that the PMS134 mutant could exert a robust dominant negative effect, resulting in biochemical and genetic manifestations of MMR deficiency. A brief description of the methods are provided below.

[0100] A hallmark of MMR deficiency is the generation of unstable microsatellite repeats in the genome of host cells. This phenotype is referred to as microsatellite instability (MI). MI consists of deletions and/or insertions within repetitive mono-, di- and/or tri nucleotide repetitive sequences throughout the entire genome of a host cell. Extensive genetic analysis eukaryotic cells have found that the only biochemical defect that is capable of producing MI is defective MMR. In light of this unique feature that defective MMR has on promoting MI, it is now used as a biochemical marker to survey for lack of MMR activity within host cells.

[0101] A method used to detect MMR deficiency in eukaryotic cells is to employ a reporter gene that has a polynucleotide repeat inserted within the coding region that disrupts its reading frame due to a frame shift. In the case where MMR is defective, the reporter gene will acquire random mutations (i.e. insertions and/or deletions) within the polynucelotide repeat yielding clones that contain a finctional reporter gene. An example of the ability to alter desired genes via defective MMR comes from experiments using Syrian Hamster fibroblasts (TK) cells (described above), where a mammalian expression construct containing a defective β-galactosidase gene (referred to as pCAR-OF) was transfected into TKPMS134 or TKvect cells as described above. The pCAR-OF vector consists of a β-galactosidase gene containing a 29-basepair poly-CA tract inserted at the 5′ end of its coding region, which causes the wild-type reading frame to shift out-of-frame. This chimeric gene is cloned into the pCEP4, which contains the constitutively cytomegalovirus (CMV) promoter upstream of the cloning site and also contains the hygromycin-resistance (HYG) gene that allows for selection of cells containing this vector. The pCAR-OF reporter cannot generate β-galactosidase activity unless a frame-restoring mutation (i.e., insertion or deletion) arises following transfection into a host. Another reporter vector called pCAR-IF contains a β-galactosidase in which a 27-bp poly-CA repeat was cloned into the same site as the pCAR-OF gene, but it is biologically active because the removal of a single repeat restores the open reading frame and produces a functional chimeric β-galactosidase polypeptide (not shown). The PCAR vectors also contain the neomycin resistance gene as selectable marker. In these proof-of-concept studies, TKPMS134 and TKvect cells were transfected with the pCAR-OF reporter vector and selected for 17 days in neomycin plus hygromycin selection medium. After the 17^(th) day, resistant colonies were stained for β-galactosidase production to determine the number of clones containing a genetically altered β-galactosidase gene. All conditions produced a relatively equal number of neomycin/hygromycin resistant cells, however, only the cells expressing the PMS134 dominant negative allele (TKPMS134) contained a subset of clones that were positive for β-galactosidase activity (Table 1). This result was also observed using a similar experimental strategy with a MMR proficient human cell line (data not shown). Table 1 shows the data from these experiments, where cell colonies were stained in situ for β-galactosidase activity and scored for activity. Cells were scored positive if the colonies turned blue in the presence of X-gal substrate and scored negative if colonies remained white. Analysis of triplicate experiments showed a significant increase in the number of β-galactosidase positive cells in the TKPMS134 cultures, while no β-galactosidase cells were seen in the control TKvect cells. TABLE 1 Number of TKPMS134 and TKvect cells containing functional β-galactosidase gene as a result of MMR deficiency. % Clones Cells White Colonies Blue Colonies with altered B-gal TKvect 65 +/− 9  0  0/65 = 0% TKPMS134 40 +/− 12 28 +/− 4 28/68 = 41%

[0102] Table 1. β-galactosidase expression of HBvec, HBPMS2 and HB 134 cells transfected with pCAR-OF reporter vectors. Cells were transfected with the pCAR-OF β-galactosidase reporter plasmid. Transfected cells were selected in hygromycin and G418, expanded and stained with X-gal solution to measure for β-galaciosidase activity (blue colored cells). 3 plates each were analyzed by microscopy. The results below represent the mean±standard deviation of these experiments.

[0103] TKPMS134/pCAR-OF clones that were pooled and expanded also showed a number of cells that contained a functional β-galactosidase gene. No β-galactosidase positive cells were observed in TKvect cells transfected with the pCAR-OF vector. These data are shown in FIG. 1 where the dark staining in panel B represent β-galactosidase positive cells present in the TKPMS134/pCAR-OF cultures while none are found in the TKvect cells grown under similar conditions (panel A). These data demonstrate the ability of dominant negative alleles of MMR genes to generate in vivo gene alterations, which allows for the rapid screening of clones with altered polypeptides exhibiting new biochemical features.

[0104] To confirm that alterations within the nucleotide sequences of the P-galactosidase gene was indeed responsible for the in vivo β-galactosidase activity present in TKPMS134 clones, RNA was isolated from TKPMS134/pCAR-OF and TKvect/pCAR-OF and the β-galactosidase mRNA primary structure was examined by reverse transcriptase polymerase chain reaction (RT-PCR) amplification and sequencing. Sequence analysis of β-galactosidase message from TKvect cells found no structural alterations in the input gene sequence. Analysis of the β-galactosidase message from TKPMS134 cells found several changes within the coding sequences of the gene. These sequence alterations included insertion and deletions of the poly-CA tract in the amino terminus as expected. Other alterations included insertions of sequences outside of the poly-CA repeat as well as a series of single base alterations (transversions and transitions) contained throughout the length of the gene.

[0105] In Situ X-Gal Staining

[0106] For in situ analysis, 100,000 cells are harvested and fixed in 1% gluteraldehyde, washed in phosphate buffered saline solution and incubated in 1 ml of X-gal substrate solution [0.15 M NaCl, 1 mM MgCl₂, 3.3 mM K₄Fe(CN)₆, 3.3 mM K₃Fe(CN)₆, 0.2% X-Gal] in 24 well plates for 2 hours at 37° C. Reactions are stopped in 500 mM sodium bicarbonate solution and transferred to microscope slides for analysis. Three plates each are counted for blue (β-galactosidase positive cells) or white (β-galactosidase negative cells) to assess for MMR inactivation. Table 1 shows the results from these studies.

EXAMPLE 2 Generation of an Expression Cassette for Screening of Structurally Altered Polypeptides in MMR Defective Cells

[0107] In order to produce recombinant proteins for screening of highly antigenic polypeptides, a fusion gene cassette was engineered that encodes for a secreted polypeptide containing a six polyhistidine domain at the C-terminus, which is useful for purification. This gene cassette is referred to as sec-hist. This gene was constructed by PCR using DNA from the pUC18 plasmid as template. The sense primers contained nucleotide sequences corresponding to the leader sequence of human interleukin-2 (ref 32), which has been found to produce robust amounts of secreted polypeptides from TK cells (personal observation). This domain was introduced at the 5′ end of the pUC18 polylinker. Antisense primers containing nucleotide sequences encoding for 6 histidines were used to position these residues at the 3′ end of the pUC18 polylinker. The nucleotide sequence of these primers are listed below. 5′aagctt ccatgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacaaacagtgca (SEQ ID NO:1) CAAAAGCTGGAGCTC-3′

[0108] The italic sequence represents a HindIII site for subcloning. The underlined sequence represents leader sequence from the human IL-2. Sequence in capital letters represents sequence from the start of the polylinker region of pUC18.

[0109] Antisense Primer:

[0110] 5′ccggatccctactagtggtgatggtgatggtgGCTTGATATCGAATTCCTG-3′ (SEQ ID NO:2)

[0111] The italic sequence represents a HindIII site for subcloning. The underlined sequence represents 6 codons encoding for histidine residues followed by 2 termination codons. Sequence in capital letters represents sequence to the 3′ end of the pUC 18 polylinker.

[0112] Amplified products were obtained using buffer conditions as previously described. Amplification reactions were carried out at 94° C. for 30 sec, 52° C. for 2 min, and 72° C. for 2 min for 25 cycles. Products were run on 1% agarose gels containing ethidium bromide, and products of the expected molecular weight were excised and purified by Gene Clean (Bio101). Products were then cloned into T-tailed vectors (InVitrogen) as suggested by the manufacturer. Recombinant clones were analyzed by restriction analysis and by DNA sequencing. Several clones contained fragments with the expected genomic sequence. The parental clone is referred to as TAsec-hist.

[0113] A schematic diagram of the sec-hist fusion protein is shown in FIG. 3A In order to generate TK cells that secrete the sec-hist polypeptide, the TAsec-hist plasmid is digested with HindIII to release the sec-hist insert. The insert was cloned into the unique HindIII site of the pCEP4 mammalian expression vector, which also contains the Hygromycin resistance gene as selectable marker. Recombinant clones were analyzed by restriction digest and sequencing to assure the authenticity of the construct.

[0114] Inserts can now be designed via PCR or direct cloning using the restricition sites contained within the polylinker (see FIG. 3B).

[0115] Recombinant pCEPsec-hist plasmid will then be transfected into TK cells as previously described using cationic lipids. Cells will be cotransfected along with the pEFPMS134, which is a mammalian expression vector containing the PMS134 dominant negative MMR gene allele under control of the constitutive elongation factor (EF) promoter. This vector contains the neomycin resistance gene and allows for double selection of TK cells for both the sec-hist and pEFPMS134 vectors. TK cells will also be cotransfected with the sec-hist and pEF empty vector as a control.

[0116] Cells are co-selected for 14 days in 0.6 mg/ml G418 and 0.8 mg/ml hygromycin B (these concentrations have been previously determined for double transfection of TK cells). After 14 days, macroscopic colonies will be isolated and subcloned into 24 well dishes (Nunc) as 1 ml cultures. Clones will then be analyzed for secreted sec-hist protein using both ELISA and western blot analysis of conditioned supernatants from sec-hist/pEFPMS134, sec-hist/pEFempty vector, and parental TK cells. A monoclonal anti-HIS antibody (Santa Cruz), which has been successfully used for other western and ELISA studies, will be used for both assays. Analysis of PMS134 expression will be determined by western blot using a PMS2-specific polyclonal antibody (Morphotek, personal communication).

[0117] ELISA will be performed on conditioned medium (CM) from TK cells transfected with pCEP4sec-hist to screen for high producers of the sec-hist polypeptide. ELISAs are carried out as follows. Twohundred microliter aliquots of conditioned medium are taken from pCEP4sec-hist transfected and control cells. Aliquots are placed into 1.5 ml eppendorf tubes and centrifuged at 14,00×g for 3 minutes to pellet cell debris. Supernates are then collected and 50 μls are placed into triplicate wells of a 96-well polystyrene microtiter plate (Nunc). Plates are incubated at room temperature for 4 hours, washed twice with 200 μls of 1× Phosphate Buffered Saline (PBS) solution, pH 7.0 (Life Technologies), and blocked with 100 μls of 5% milk in 1×PBS for 1 hour. Plates are then incubated with a monoclonal anti-HIS antibody (diluted 1:1000 in 1×PBS) (Santa Cruz) for 2 hours at room temperature and then washed twice with 200 μls of 1×PBS, and probed with an anti-mouse-horse radish peroxidase (HRP) conjugated secondary antibody diluted 1:3000 in PBS. Plates are then incubated at room temperature for 1 hour, washed three times with 200 μls of 1×PBS and incubated with TMB substrate (BioRad) for 15-30 minutes. After incubation is completed, reactions are stopped using 0.1N H₂SO₄ and plates are read using a BioRad microplate reader at 415 nm. Clones are determined to be positive for secreted sec-hist if expected cells are found to produce a significant signal over control cells. Conditioned medium from positive cultures will then be analyzed by western blot using the anti-HIS antibody as probe to confirm ELISA data.

[0118] Western blot analysis will be carried out as follows. Briefly, 50 μs of CM or 50,000 cells from each culture is directly lysed in 2× lysis buffer (60 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 0.1 M 2-mercaptoethanol, 0.001% bromophenol blue) and samples are boiled for 5 minutes. Lysate proteins are separated by electrophoresis on 4-20% Tris glycine gels (Novex) and then electroblotted onto Immobilon-P (Millipore) in 48 mM Tris base, 40 mM glycine, 0.0375% SDS, 20% methanol and blocked overnight at 4° C. in Tris-buffered saline plus 0.05% Tween-20 and 5% condensed milk. Filters are then probed with an antibody generated against the PMS134 or the polyHIS tag (Santa Cruz), followed by a secondary HRP-conjugated antibody. After incubation with the secondary antibody, blots are developed using chemiluminescence (Pierce) and exposed to film to determine PMS134 and sec-hist expression. Clones exhibiting expression of both genes will then be used in experiments described above.

[0119] A potential technical problem may exist in expressing the sec-hist protein due to toxicity that it may have on the growth of TK cells. If the production of no or low amounts of sec-hist polypeptide is found to occur in the above analysis, a HindIII sec-hist fragment from the TAsec-hist plasmid will be subcloned into the unique HindIII site of the pND/V5 steroid inducible vector (Invitrogen). This vector has been found to produce robust protein expression in TK cells upon in steroid induction. This application teaches the use of employing an inducible vector containing the sec-hist expression cassette to express polypeptides in TK cells that may be toxic under constitutively expressed conditions. Cells that are found to co-express the PMS134 and the sec-hist genes or the control cell expressing the pEF empty vector and the sec-hist gene are cultured under high growth conditions in media containing neomycin, hygromycin and vitamins, which has been shown to increase the doubling time of TK cells and enhance the genetic alteration of β-galactosidase reporter plasmids in vivo (data not shown). Briefly, cells are grown in vitamin enriched medium for 20 doublings (˜17 days), a time at which it has been found that 20-40% of clones contain sequence alterations within a particular genetic locus. After selection, cells will be subcloned in 96-well microtiter plates by limiting dilutions. Clones will be grown for 5 days in the presence of neomycin/hygromycin-free medium containing heat inactivated serum to remove complement for in vitro antigenic assays that will be performed using murine lymphocytes as described in section below.

[0120] The sec-hist expression vector cassette can also be transfected into cell lines that are “naturally” defective for MMR such as the human cell lines derived from colon cancer tumors such as but not limited to HCT116 and DLD-1. The vector can be in the constitutive backbone pCEP4 or under control of the steroid-inducible vectors pIND or pMAM.

[0121] The techniques described above teach us the use of producing structurally altered antigens from mammalian cells to ensure proper folding or post-translation modifications of the polypeptide. This approach gives an advantage over others that employ the use of prokaryotic, yeast or baculovirus produced antigens that have been found to produce weak antigenic responses due to misfolding or improper post-translational modifications.

EXAMPLE 3 Screening Strategy to Identify Cell Clones Producing Highly Antigenic Polypeptides

[0122] In order to identify antigenic polypeptides produced by TKPMS134 cell clones, the following in vitro assays will be performed.

[0123] First, the lymphocyte stimulatory activity of sec-hist polypeptides will be measured by adding CM of TKsec-hist cells with or without the PMS134 to lymphocytes derived from naive or whole antigen exposed Balb/C mice. Briefly, 2 mice will be infected with whole antigen in the presence of Freund's Complete Adjuvant by subcutaneous injection in the tail with a 100-μl 1/1 mixture of complete Freund's adjuvant (CFA) (Difco). Two subcutaneous boosts will be performed with the same quantity of antigen, mixed 1/1 with incomplete Freund's adjuvant (Difco), after 2 and 4 weeks. Two control mice will receive adjuvant alone. Mice are sacrificed 5 days after the second boost (at day 33). Peripheral blood mononuclear cells (PBMCs) from whole blood and splenocytes from spleens of will be harvested following the previously described procedures (Nicolaides, N. C. et al. (1997) Proc. Natl. Acad. Sci. USA 94:13175-13180). For splenocyte assays, whole spleens are pressed through sterile wire mesh into RPMI medium (Life Technology). Next, cells are washed twice in RPMI and incubated for 10 minutes in RBC lysis buffer. Cells are then washed again and resuspended at 1×10⁵ cells/ml in RPMI-1640 medium plus 10% heat inactivated fetal bovine serum. One hundred microliters of cells are aliquoted into twenty 96-well titer flat bottom plates.

[0124] For PBMC isolation, whole blood is isolated by eye puncture and collected into vacutainer tubes containing EDTA. An equal volume of PBS (Mg²⁺/Ca²⁺-free) is added to whole blood. PBMCs are isolated by centrifugation over Ficoll-Paque gradients (Pharmacia Biotech 17-1440-02). Purified cells are seeded at 1×10⁵ cells/ml in RPMI 1640 containing 10% heat-inactivated fetal bovine serum (Life Technologies, Inc.) and 100 μls are plated in 96 well flat bottom microtiter plates and incubated at 37° C. in 5% CO₂.

[0125] To measure for T-cell activation, PBMCs and splenocytes from primed and non-primed mice are incubated with 10% conditioned medium (CM) from TKsec-hist cells with or without the PMS134 gene. 5 μg/ml of concavalinA (ConA) is used as a positive control for splenocyte culture assays, while 5 μg/ml phorbol 12-myristate 13-acetate and 1 μg/ml phytohemagglutinin (Sigma) are used as a positive control for PBMC cultures. CM from parental TK cells grown in the presence of RPMI with 10% heat inactivated medium will be used as negative control. Previous studies using CM from TK cells have found no stimulatory activity to be produced on PBMCs or splenocytes (N. Nicolaides, personal observation). Cultures are incubated at 37° C. in 5% CO₂ for 6 days and scored for antigenic activity as determined by proliferation assay. Proliferation is assayed using a modified protocol of the acid phosphatase assay as described (Grasso, L. et al. (1998) J. Biol. Chem. 273:24016-24024). Briefly, 50 μls of a buffer containing 0.1 M sodium acetate (pH 5.5), 0.1% Triton X-100, and 10 mM p-nitrophenyl phosphate (Sigma 104 phosphatase substrate) is added directly to each well containing 0.2 ml of growth medium and incubated for 1.5 h at room temperature. Reactions are terminated by the addition of 0.05 N sodium hydroxide and quantified by absorbance at 410 nm using a BioRad plate reader. Data is represented as a stimulation index (SI), which is the proliferation of experimental data points divided by the mean of 10 aliquots of CM from TK parental cells. All experiments will be performed in at least triplicate.

[0126] It is expected that several TKsec-hist clones co-expressing the PMS134 protein will be found to have an enhanced antigenicity on PBMCs and/or splenocytes due to conformational changes that will occur within the coding region of the target antigen. These changes may form secondary domains that serve as T and/or B cell epitopes and in turn are responsible for stimulating their respective activation. Clones that reproducibly produce enhanced antigenic activation will be sequenced to confirm and identify that a structural alteration(s) has indeed occurred within the coding region of the gene. Sequence data may also shed additional light into the importance of critical domains within this candidate vaccine polypeptide for additional rounds of alteration that may lead to the creation of a super-antigen that may serve as a potent vaccine.

[0127] Sequence analysis of clones will be performed as follows. First positive clones will be expanded from 96-well plates to 24-well plates. Confluent wells will be expanded and cells will be harvested for RNA extraction. RNA extraction and reverse transcription will be carried out using the Trizol method as previously described (Nicolaides, N. C. et al. (1997) Proc. Natl. Acad. Sci. USA 94:13175-13180; Grasso, L. et al. (1998) J. Biol. Chem. 273:24016-24024). Reverse transcription will be carried out using Superscript II (Life Technology) as previously described (Nicolaides, N. C. et al. (1998) Mol. Cell. Biol. 18:1635-1641). cDNAs will be amplified to isolate the sec-hist transcript using the sense primer: 5′-catgtacaggatgcaactcctg-3′ (SEQ ID NO:3), which is located at the IL-2 leader sequence site (see FIG. 3), and the antisense primer: 5′-tactagtggtgatggtgatggtg-3′ (SEQ ID NO:4), which is located at the C-terminal polyhis site. Amplification is carried out at 94° C. for 30 sec, 52° C. for 2 min, 72° C. for 2 min for 30 cycles. Reactions are analyzed on agarose gels. If products of the expected molecular weight are generated then samples will be cleaned using the QIAquick PCR template kit (Qiagen) to remove PCR amplimers and sequenced using the following primers that cover the entire coding region of the sec-hist gene. Clones are then sequenced using primers specific to the gene encoding the antigen.

[0128] Clones producing genetically altered sec-hist polypeptides will then be expanded into T-75 flasks to a density that will enable for the sufficient production of secreted sec-hist polypeptide in the CM. Conditioned medium containing the sec-hist polypeptide is then collected, and centrifuged at 3,500×g for 10 minutes to remove cellular debris. CM is then loaded onto a 10 ml-HiTrap Nickel column following the manufacturer's protocol (Pharmacia). After absorption and washing, the column is treated with 200 mM imidazole to elute the fusion protein. Recovered polypeptides are then analyzed by SDS-PAGE using 4-12% NuPAGE gels (Novex) and silver stained following the manufacturer's protocol (Novex). Due to the random nature of defective MMR, the possibility exists that sec-hist alleles may be generated by clones producing enhanced antigenic polypeptides, which contain a nonsense or frame-shift mutation, therefore forming polypeptides lacking a polyHIS C-terminus. If a nonsense or frameshift mutation does occur in clones producing polypeptides with increased antigenicity, then the new allele is reengineered via PCR to contain a polyHIS tag at the C-terminus, and this new fusion protein will be rescreened as above.

[0129] Purified polypeptides will be rescreened at a final concentration of 10 μg/ml in the in vitro assays described above to confirm that the antigenic activity is indeed coming from the sec-hist protein. TK cells producing altered antigens with enhanced activity on both PBMCs and splenocytes will then be tested along with the non-altered sec-hist protein in vivo in Balb/C mice for the ability to illicit an immune response in the absence of adjuvant. A schematic diagram outlining the screening procedure is given in FIG. 4.

EXAMPLE 4 Screening Strategy to Identify Cell Clones Producing Highly Immunogenic Antigens

[0130] To test the immunogenic potential that the altered polypeptides identified from EXAMPLE 3 have in vivo, Balb/C mice will be injected with the 6 most antigenic polypeptides and the wild type sec-hist polypeptide produced from TKEFempty/sec-hist cells in the absence of adjuvant. Briefly, mice will be immunized with 30 μgs of purified sec-hist protein in sterile phosphate buffered saline (PBS) without adjuvant by subcutaneous injection in the tail. One group of mice will receive a 100 μls of a mixture of polypeptides diluted 1/1 in complete Freund's adjuvant (CFA) (Difco) as a positive control. Two subcutaneous boosts will be performed with the same quantity of antigen, and the mouse receiving the polypeptide mixture with adjuvant will be boosted with a 1/1 mixture with incomplete Freund's adjuvant (Difco), at 2 and 4 weeks after the initial injection. Mice will be bled 5 and 15 days after the second boost (at day 33) to measure for antigen titers. Control mice will receive PBS alone. Before the start of immunization, a prebleed will be obtained from each mouse.

[0131] Immune responses will be measured by screening for whole Ig and sec-hist specific antibody titers by ELISA following the protocol described above. For antigen specific antisera titers, 96-well plates will be coated with a 50 uls of a solution containing 5 μg/ml each antigen used for vaccination in PBS. Plates will then be probed with serial dilutions of prebleeds, 5 day and 15 day bleeds. Detection of antibodies will be done using a sheep anti-mouse-HRP conjugated secondary antibody followed by incubation with TMB substrate as described above.

[0132] Success of the strategy proposed in this program will be demonstrated with the generation of antisera from mice immunized with altered sec-hist protein that is able to cross react with the native sec-hist protein. If no titers are found in the samples without adjuvant, mice will be administrated antigen in complete Freund's Adjuvant and titers analyzed 14 days later, followed by immunization in Incomplete Freund's at day 17 if no titers are found and analyzed at day 31.

[0133] A potential problem that may occur with the outlined strategy is that antibody titers may be generated against the sec-polyhistidine fusion domain. To determine if antisera is able to identify authentic sec-his polypeptides, the antigen lacking the polyhistidine domain will be generated by in vitro transcription-translation (TNT) in the presence of radiolabelled methionine. A template containing the sec-hist polypeptide will also be made and the encoded protein used as a control. Templates for the TNT reactions will be generated by PCR as described (Nicolaides, N. C. et al. (1998) Mol. Cell. Biol. 18:1635-1641). Briefly, the TAsec-hist plasmid will be used as template to amplify gene segments that encode for the untqagged antigen or the sec-hist tagged protein.

[0134] Translated polypeptides are first analyzed by SDS-PAGE gel electrophoresis and autoradiography to ensure that the polypeptide with the expected molecular weight are synthesized. Proteins are then immunoprecipitated using antiserum from vaccinated mice to determine if these antibodies recognize authentic antigen sequences. Briefly, immunoprecipitations are performed on in vitro-translated proteins by mixing the translation reaction mixtures with 100 μls of mouse antiserum or 1 μg of a HIS-specific monoclonal antibody (MAB) (Santa Cruz) in 400 μls of EBC buffer (50 mM Tris [pH 7.5], 0.1 M NaCl, 0.5% Nonidet P-40). After incubation for 1 h at 4° C., protein A-Sepharose (Sigma) is added to a final concentration of 10% and the reaction mixtures are incubated at 4° C. for 1 h. Proteins bound to protein A are washed five times in EBC and separated by electrophoresis on 4-20% Tris-glycine gradient gels, which are then dried and autoradiographed.

[0135] If antisera are able to react with authentic sequences lacking HIS residues, the data from these studies will be continued in further preclinical animal studies.

[0136] Discussion

[0137] The initial steps of MMR are dependent on two protein complexes, called MutScα and MutLα. The use of dominant negative MMR alleles are able to perturb the formation of these complexes with downstream biochemicals involved in the excision and polymerization of nucleotides comprising the “corrected” nucleotides. Examples from this application show the ability of a truncated MMR allele (PMS134 is capable of blocking MMR resulting in a hypermutable cell line that gains genetic alterations throughout its entire genome per cell division. Once a cell line is produced that contains genetic alterations within genes encoding for an antigen, it is desirable to restore the genomic integrity of the cell host. This can be achieved by the use of inducible vectors whereby dominant negative MMR genes are cloned into such vectors, introduced into antigen-producing cells and the cells are cultured in the presence of inducer molecules and/or conditions. Inducible vectors include but are not limited to chemical regulated promoters such as the steroid inducible MMTV, tetracycline regulated promoters, temperature sensitive MMR gene alleles, and temperature sensitive promoters.

[0138] The results described above lead to several conclusions. First, expression of PMS134 results in an increase in microsattelite instability in TK cells. That this elevated microsattelite instability is due to MMR deficiency was proven by evaluation of extracts from stably transduced cells and stability of a tract contained within the pCAR-OF vector. The expression of PMS134 results in a polar defect in MMR, which was only observed using heteroduplexes designed to test repair from the 5′ direction (no significant defect in repair from the 3′ direction was observed in the same extracts). Interestingly, cells deficient in hMLH1 also have a polar defect in MMR, but in this case preferentially affecting repair from the 3′ direction. It is known from previous studies in both prokaryotes and eukaryotes that the separate enzymatic components mediate repair from the two different directions. These results strongly suggest a model in which 5′ repair is primarily dependent on hPMS2 while 3′ repair is primarily dependent on hMLH1. It is easy to envision how the dimeric complex between PMS2 and MLH1 might set up this directionality. The combined results also demonstrate that a defect in directional MMR is sufficient to produce a MMR defective phenotype and suggests that any MMR gene allele is useful to produce genetically altered TK cells, or a cell line that is producing antigenic gene products. Moreover, the use of such MMR alleles will be useful for generating genetically altered polypeptides with altered structures as effective vaccine agents.

[0139] This application also teaches us that ANY method used to block MMR can be performed to generate hypermutablility in an antigen-producing cell that can lead to genetically altered proteins with enhanced biochemical features such as but not limited to increased antigenicity, increased immunogenicity, and enhanced pharmacokinetic profiles.

[0140] The blockade of MMR in such cells can be through the use of dominant negative MMR gene alleles from any species including bacteria, yeast, protozoa, insects, rodents, primates, mammalian cells, and man. Blockade of MMR can also be generated through the use of antisense RNA or deoxynucleotides directed to any of the genes involved in the MMR biochemical pathway. Blockade of MMR can be through the use of polypeptides that interfere with subunits of the MMR complex including but not limited to antibodies. Finally, the blockade of MMR may be through the use chemicals such as but not limited to nonhydrolyzable ATP analogs, which have been shown to block MMR (Galio, L. et al. (1999) Nucl. Acids Res. 27:2325-2331; Spampinato, C. and P. Modrich (2000) J. Biol. Chem. 275:9863-9869).

1 17 1 86 DNA Artificial Sequence Oligonucleotide primer 1 aagcttccat gtacaggatg caactcctgt cttgcattgc actaagtctt gcacttgtca 60 caaacagtgc acaaaagctg gagctc 86 2 51 DNA Artificial Sequence Oligonucleotide primer 2 ccggatccct actagtggtg atggtgatgg tggcttgata tcgaattcct g 51 3 22 DNA Artificial Sequence Oligonucleotide primer 3 catgtacagg atgcaactcc tg 22 4 23 DNA Artificial Sequence Oligonucleotide primer 4 tactagtggt gatggtgatg gtg 23 5 859 PRT Mus musculus 5 Met Glu Gln Thr Glu Gly Val Ser Thr Glu Cys Ala Lys Ala Ile Lys 1 5 10 15 Pro Ile Asp Gly Lys Ser Val His Gln Ile Cys Ser Gly Gln Val Ile 20 25 30 Leu Ser Leu Ser Thr Ala Val Lys Glu Leu Ile Glu Asn Ser Val Asp 35 40 45 Ala Gly Ala Thr Thr Ile Asp Leu Arg Leu Lys Asp Tyr Gly Val Asp 50 55 60 Leu Ile Glu Val Ser Asp Asn Gly Cys Gly Val Glu Glu Glu Asn Phe 65 70 75 80 Glu Gly Leu Ala Leu Lys His His Thr Ser Lys Ile Gln Glu Phe Ala 85 90 95 Asp Leu Thr Gln Val Glu Thr Phe Gly Phe Arg Gly Glu Ala Leu Ser 100 105 110 Ser Leu Cys Ala Leu Ser Asp Val Thr Ile Ser Thr Cys His Gly Ser 115 120 125 Ala Ser Val Gly Thr Arg Leu Val Phe Asp His Asn Gly Lys Ile Thr 130 135 140 Gln Lys Thr Pro Tyr Pro Arg Pro Lys Gly Thr Thr Val Ser Val Gln 145 150 155 160 His Leu Phe Tyr Thr Leu Pro Val Arg Tyr Lys Glu Phe Gln Arg Asn 165 170 175 Ile Lys Lys Glu Tyr Ser Lys Met Val Gln Val Leu Gln Ala Tyr Cys 180 185 190 Ile Ile Ser Ala Gly Val Arg Val Ser Cys Thr Asn Gln Leu Gly Gln 195 200 205 Gly Lys Arg His Ala Val Val Cys Thr Ser Gly Thr Ser Gly Met Lys 210 215 220 Glu Asn Ile Gly Ser Val Phe Gly Gln Lys Gln Leu Gln Ser Leu Ile 225 230 235 240 Pro Phe Val Gln Leu Pro Pro Ser Asp Ala Val Cys Glu Glu Tyr Gly 245 250 255 Leu Ser Thr Ser Gly Arg His Lys Thr Phe Ser Thr Phe Arg Ala Ser 260 265 270 Phe His Ser Ala Arg Thr Ala Pro Gly Gly Val Gln Gln Thr Gly Ser 275 280 285 Phe Ser Ser Ser Ile Arg Gly Pro Val Thr Gln Gln Arg Ser Leu Ser 290 295 300 Leu Ser Met Arg Phe Tyr His Met Tyr Asn Arg His Gln Tyr Pro Phe 305 310 315 320 Val Val Leu Asn Val Ser Val Asp Ser Glu Cys Val Asp Ile Asn Val 325 330 335 Thr Pro Asp Lys Arg Gln Ile Leu Leu Gln Glu Glu Lys Leu Leu Leu 340 345 350 Ala Val Leu Lys Thr Ser Leu Ile Gly Met Phe Asp Ser Asp Ala Asn 355 360 365 Lys Leu Asn Val Asn Gln Gln Pro Leu Leu Asp Val Glu Gly Asn Leu 370 375 380 Val Lys Leu His Thr Ala Glu Leu Glu Lys Pro Val Pro Gly Lys Gln 385 390 395 400 Asp Asn Ser Pro Ser Leu Lys Ser Thr Ala Asp Glu Lys Arg Val Ala 405 410 415 Ser Ile Ser Arg Leu Arg Glu Ala Phe Ser Leu His Pro Thr Lys Glu 420 425 430 Ile Lys Ser Arg Gly Pro Glu Thr Ala Glu Leu Thr Arg Ser Phe Pro 435 440 445 Ser Glu Lys Arg Gly Val Leu Ser Ser Tyr Pro Ser Asp Val Ile Ser 450 455 460 Tyr Arg Gly Leu Arg Gly Ser Gln Asp Lys Leu Val Ser Pro Thr Asp 465 470 475 480 Ser Pro Gly Asp Cys Met Asp Arg Glu Lys Ile Glu Lys Asp Ser Gly 485 490 495 Leu Ser Ser Thr Ser Ala Gly Ser Glu Glu Glu Phe Ser Thr Pro Glu 500 505 510 Val Ala Ser Ser Phe Ser Ser Asp Tyr Asn Val Ser Ser Leu Glu Asp 515 520 525 Arg Pro Ser Gln Glu Thr Ile Asn Cys Gly Asp Leu Asp Cys Arg Pro 530 535 540 Pro Gly Thr Gly Gln Ser Leu Lys Pro Glu Asp His Gly Tyr Gln Cys 545 550 555 560 Lys Ala Leu Pro Leu Ala Arg Leu Ser Pro Thr Asn Ala Lys Arg Phe 565 570 575 Lys Thr Glu Glu Arg Pro Ser Asn Val Asn Ile Ser Gln Arg Leu Pro 580 585 590 Gly Pro Gln Ser Thr Ser Ala Ala Glu Val Asp Val Ala Ile Lys Met 595 600 605 Asn Lys Arg Ile Val Leu Leu Glu Phe Ser Leu Ser Ser Leu Ala Lys 610 615 620 Arg Met Lys Gln Leu Gln His Leu Lys Ala Gln Asn Lys His Glu Leu 625 630 635 640 Ser Tyr Arg Lys Phe Arg Ala Lys Ile Cys Pro Gly Glu Asn Gln Ala 645 650 655 Ala Glu Asp Glu Leu Arg Lys Glu Ile Ser Lys Ser Met Phe Ala Glu 660 665 670 Met Glu Ile Leu Gly Gln Phe Asn Leu Gly Phe Ile Val Thr Lys Leu 675 680 685 Lys Glu Asp Leu Phe Leu Val Asp Gln His Ala Ala Asp Glu Lys Tyr 690 695 700 Asn Phe Glu Met Leu Gln Gln His Thr Val Leu Gln Ala Gln Arg Leu 705 710 715 720 Ile Thr Pro Gln Thr Leu Asn Leu Thr Ala Val Asn Glu Ala Val Leu 725 730 735 Ile Glu Asn Leu Glu Ile Phe Arg Lys Asn Gly Phe Asp Phe Val Ile 740 745 750 Asp Glu Asp Ala Pro Val Thr Glu Arg Ala Lys Leu Ile Ser Leu Pro 755 760 765 Thr Ser Lys Asn Trp Thr Phe Gly Pro Gln Asp Ile Asp Glu Leu Ile 770 775 780 Phe Met Leu Ser Asp Ser Pro Gly Val Met Cys Arg Pro Ser Arg Val 785 790 795 800 Arg Gln Met Phe Ala Ser Arg Ala Cys Arg Lys Ser Val Met Ile Gly 805 810 815 Thr Ala Leu Asn Ala Ser Glu Met Lys Lys Leu Ile Thr His Met Gly 820 825 830 Glu Met Asp His Pro Trp Asn Cys Pro His Gly Arg Pro Thr Met Arg 835 840 845 His Val Ala Asn Leu Asp Val Ile Ser Gln Asn 850 855 6 3056 DNA Mus musculus 6 gaattccggt gaaggtcctg aagaatttcc agattcctga gtatcattgg aggagacaga 60 taacctgtcg tcaggtaacg atggtgtata tgcaacagaa atgggtgttc ctggagacgc 120 gtcttttccc gagagcggca ccgcaactct cccgcggtga ctgtgactgg aggagtcctg 180 catccatgga gcaaaccgaa ggcgtgagta cagaatgtgc taaggccatc aagcctattg 240 atgggaagtc agtccatcaa atttgttctg ggcaggtgat actcagttta agcaccgctg 300 tgaaggagtt gatagaaaat agtgtagatg ctggtgctac tactattgat ctaaggctta 360 aagactatgg ggtggacctc attgaagttt cagacaatgg atgtggggta gaagaagaaa 420 actttgaagg tctagctctg aaacatcaca catctaagat tcaagagttt gccgacctca 480 cgcaggttga aactttcggc tttcgggggg aagctctgag ctctctgtgt gcactaagtg 540 atgtcactat atctacctgc cacgggtctg caagcgttgg gactcgactg gtgtttgacc 600 ataatgggaa aatcacccag aaaactccct acccccgacc taaaggaacc acagtcagtg 660 tgcagcactt attttataca ctacccgtgc gttacaaaga gtttcagagg aacattaaaa 720 aggagtattc caaaatggtg caggtcttac aggcgtactg tatcatctca gcaggcgtcc 780 gtgtaagctg cactaatcag ctcggacagg ggaagcggca cgctgtggtg tgcacaagcg 840 gcacgtctgg catgaaggaa aatatcgggt ctgtgtttgg ccagaagcag ttgcaaagcc 900 tcattccttt tgttcagctg ccccctagtg acgctgtgtg tgaagagtac ggcctgagca 960 cttcaggacg ccacaaaacc ttttctacgt ttcgggcttc atttcacagt gcacgcacgg 1020 cgccgggagg agtgcaacag acaggcagtt tttcttcatc aatcagaggc cctgtgaccc 1080 agcaaaggtc tctaagcttg tcaatgaggt tttatcacat gtataaccgg catcagtacc 1140 catttgtcgt ccttaacgtt tccgttgact cagaatgtgt ggatattaat gtaactccag 1200 ataaaaggca aattctacta caagaagaga agctattgct ggccgtttta aagacctcct 1260 tgataggaat gtttgacagt gatgcaaaca agcttaatgt caaccagcag ccactgctag 1320 atgttgaagg taacttagta aagctgcata ctgcagaact agaaaagcct gtgccaggaa 1380 agcaagataa ctctccttca ctgaagagca cagcagacga gaaaagggta gcatccatct 1440 ccaggctgag agaggccttt tctcttcatc ctactaaaga gatcaagtct aggggtccag 1500 agactgctga actgacacgg agttttccaa gtgagaaaag gggcgtgtta tcctcttatc 1560 cttcagacgt catctcttac agaggcctcc gtggctcgca ggacaaattg gtgagtccca 1620 cggacagccc tggtgactgt atggacagag agaaaataga aaaagactca gggctcagca 1680 gcacctcagc tggctctgag gaagagttca gcaccccaga agtggccagt agctttagca 1740 gtgactataa cgtgagctcc ctagaagaca gaccttctca ggaaaccata aactgtggtg 1800 acctggactg ccgtcctcca ggtacaggac agtccttgaa gccagaagac catggatatc 1860 aatgcaaagc tctacctcta gctcgtctgt cacccacaaa tgccaagcgc ttcaagacag 1920 aggaaagacc ctcaaatgtc aacatttctc aaagattgcc tggtcctcag agcacctcag 1980 cagctgaggt cgatgtagcc ataaaaatga ataagagaat cgtgctcctc gagttctctc 2040 tgagttctct agctaagcga atgaagcagt tacagcacct aaaggcgcag aacaaacatg 2100 aactgagtta cagaaaattt agggccaaga tttgccctgg agaaaaccaa gcagcagaag 2160 atgaactcag aaaagagatt agtaaatcga tgtttgcaga gatggagatc ttgggtcagt 2220 ttaacctggg atttatagta accaaactga aagaggacct cttcctggtg gaccagcatg 2280 ctgcggatga gaagtacaac tttgagatgc tgcagcagca cacggtgctc caggcgcaga 2340 ggctcatcac accccagact ctgaacttaa ctgctgtcaa tgaagctgta ctgatagaaa 2400 atctggaaat attcagaaag aatggctttg actttgtcat tgatgaggat gctccagtca 2460 ctgaaagggc taaattgatt tccttaccaa ctagtaaaaa ctggaccttt ggaccccaag 2520 atatagatga actgatcttt atgttaagtg acagccctgg ggtcatgtgc cggccctcac 2580 gagtcagaca gatgtttgct tccagagcct gtcggaagtc agtgatgatt ggaacggcgc 2640 tcaatgcgag cgagatgaag aagctcatca cccacatggg tgagatggac cacccctgga 2700 actgccccca cggcaggcca accatgaggc acgttgccaa tctggatgtc atctctcaga 2760 actgacacac cccttgtagc atagagttta ttacagattg ttcggtttgc aaagagaagg 2820 ttttaagtaa tctgattatc gttgtacaaa aattagcatg ctgctttaat gtactggatc 2880 catttaaaag cagtgttaag gcaggcatga tggagtgttc ctctagctca gctacttggg 2940 tgatccggtg ggagctcatg tgagcccagg actttgagac cactccgagc cacattcatg 3000 agactcaatt caaggacaaa aaaaaaaaga tatttttgaa gccttttaaa aaaaaa 3056 7 862 PRT Homo sapiens 7 Met Glu Arg Ala Glu Ser Ser Ser Thr Glu Pro Ala Lys Ala Ile Lys 1 5 10 15 Pro Ile Asp Arg Lys Ser Val His Gln Ile Cys Ser Gly Gln Val Val 20 25 30 Leu Ser Leu Ser Thr Ala Val Lys Glu Leu Val Glu Asn Ser Leu Asp 35 40 45 Ala Gly Ala Thr Asn Ile Asp Leu Lys Leu Lys Asp Tyr Gly Val Asp 50 55 60 Leu Ile Glu Val Ser Asp Asn Gly Cys Gly Val Glu Glu Glu Asn Phe 65 70 75 80 Glu Gly Leu Thr Leu Lys His His Thr Ser Lys Ile Gln Glu Phe Ala 85 90 95 Asp Leu Thr Gln Val Glu Thr Phe Gly Phe Arg Gly Glu Ala Leu Ser 100 105 110 Ser Leu Cys Ala Leu Ser Asp Val Thr Ile Ser Thr Cys His Ala Ser 115 120 125 Ala Lys Val Gly Thr Arg Leu Met Phe Asp His Asn Gly Lys Ile Ile 130 135 140 Gln Lys Thr Pro Tyr Pro Arg Pro Arg Gly Thr Thr Val Ser Val Gln 145 150 155 160 Gln Leu Phe Ser Thr Leu Pro Val Arg His Lys Glu Phe Gln Arg Asn 165 170 175 Ile Lys Lys Glu Tyr Ala Lys Met Val Gln Val Leu His Ala Tyr Cys 180 185 190 Ile Ile Ser Ala Gly Ile Arg Val Ser Cys Thr Asn Gln Leu Gly Gln 195 200 205 Gly Lys Arg Gln Pro Val Val Cys Thr Gly Gly Ser Pro Ser Ile Lys 210 215 220 Glu Asn Ile Gly Ser Val Phe Gly Gln Lys Gln Leu Gln Ser Leu Ile 225 230 235 240 Pro Phe Val Gln Leu Pro Pro Ser Asp Ser Val Cys Glu Glu Tyr Gly 245 250 255 Leu Ser Cys Ser Asp Ala Leu His Asn Leu Phe Tyr Ile Ser Gly Phe 260 265 270 Ile Ser Gln Cys Thr His Gly Val Gly Arg Ser Ser Thr Asp Arg Gln 275 280 285 Phe Phe Phe Ile Asn Arg Arg Pro Cys Asp Pro Ala Lys Val Cys Arg 290 295 300 Leu Val Asn Glu Val Tyr His Met Tyr Asn Arg His Gln Tyr Pro Phe 305 310 315 320 Val Val Leu Asn Ile Ser Val Asp Ser Glu Cys Val Asp Ile Asn Val 325 330 335 Thr Pro Asp Lys Arg Gln Ile Leu Leu Gln Glu Glu Lys Leu Leu Leu 340 345 350 Ala Val Leu Lys Thr Ser Leu Ile Gly Met Phe Asp Ser Asp Val Asn 355 360 365 Lys Leu Asn Val Ser Gln Gln Pro Leu Leu Asp Val Glu Gly Asn Leu 370 375 380 Ile Lys Met His Ala Ala Asp Leu Glu Lys Pro Met Val Glu Lys Gln 385 390 395 400 Asp Gln Ser Pro Ser Leu Arg Thr Gly Glu Glu Lys Lys Asp Val Ser 405 410 415 Ile Ser Arg Leu Arg Glu Ala Phe Ser Leu Arg His Thr Thr Glu Asn 420 425 430 Lys Pro His Ser Pro Lys Thr Pro Glu Pro Arg Arg Ser Pro Leu Gly 435 440 445 Gln Lys Arg Gly Met Leu Ser Ser Ser Thr Ser Gly Ala Ile Ser Asp 450 455 460 Lys Gly Val Leu Arg Pro Gln Lys Glu Ala Val Ser Ser Ser His Gly 465 470 475 480 Pro Ser Asp Pro Thr Asp Arg Ala Glu Val Glu Lys Asp Ser Gly His 485 490 495 Gly Ser Thr Ser Val Asp Ser Glu Gly Phe Ser Ile Pro Asp Thr Gly 500 505 510 Ser His Cys Ser Ser Glu Tyr Ala Ala Ser Ser Pro Gly Asp Arg Gly 515 520 525 Ser Gln Glu His Val Asp Ser Gln Glu Lys Ala Pro Glu Thr Asp Asp 530 535 540 Ser Phe Ser Asp Val Asp Cys His Ser Asn Gln Glu Asp Thr Gly Cys 545 550 555 560 Lys Phe Arg Val Leu Pro Gln Pro Thr Asn Leu Ala Thr Pro Asn Thr 565 570 575 Lys Arg Phe Lys Lys Glu Glu Ile Leu Ser Ser Ser Asp Ile Cys Gln 580 585 590 Lys Leu Val Asn Thr Gln Asp Met Ser Ala Ser Gln Val Asp Val Ala 595 600 605 Val Lys Ile Asn Lys Lys Val Val Pro Leu Asp Phe Ser Met Ser Ser 610 615 620 Leu Ala Lys Arg Ile Lys Gln Leu His His Glu Ala Gln Gln Ser Glu 625 630 635 640 Gly Glu Gln Asn Tyr Arg Lys Phe Arg Ala Lys Ile Cys Pro Gly Glu 645 650 655 Asn Gln Ala Ala Glu Asp Glu Leu Arg Lys Glu Ile Ser Lys Thr Met 660 665 670 Phe Ala Glu Met Glu Ile Ile Gly Gln Phe Asn Leu Gly Phe Ile Ile 675 680 685 Thr Lys Leu Asn Glu Asp Ile Phe Ile Val Asp Gln His Ala Thr Asp 690 695 700 Glu Lys Tyr Asn Phe Glu Met Leu Gln Gln His Thr Val Leu Gln Gly 705 710 715 720 Gln Arg Leu Ile Ala Pro Gln Thr Leu Asn Leu Thr Ala Val Asn Glu 725 730 735 Ala Val Leu Ile Glu Asn Leu Glu Ile Phe Arg Lys Asn Gly Phe Asp 740 745 750 Phe Val Ile Asp Glu Asn Ala Pro Val Thr Glu Arg Ala Lys Leu Ile 755 760 765 Ser Leu Pro Thr Ser Lys Asn Trp Thr Phe Gly Pro Gln Asp Val Asp 770 775 780 Glu Leu Ile Phe Met Leu Ser Asp Ser Pro Gly Val Met Cys Arg Pro 785 790 795 800 Ser Arg Val Lys Gln Met Phe Ala Ser Arg Ala Cys Arg Lys Ser Val 805 810 815 Met Ile Gly Thr Ala Leu Asn Thr Ser Glu Met Lys Lys Leu Ile Thr 820 825 830 His Met Gly Glu Met Asp His Pro Trp Asn Cys Pro His Gly Arg Pro 835 840 845 Thr Met Arg His Ile Ala Asn Leu Gly Val Ile Ser Gln Asn 850 855 860 8 2771 DNA Homo sapiens 8 cgaggcggat cgggtgttgc atccatggag cgagctgaga gctcgagtac agaacctgct 60 aaggccatca aacctattga tcggaagtca gtccatcaga tttgctctgg gcaggtggta 120 ctgagtctaa gcactgcggt aaaggagtta gtagaaaaca gtctggatgc tggtgccact 180 aatattgatc taaagcttaa ggactatgga gtggatctta ttgaagtttc agacaatgga 240 tgtggggtag aagaagaaaa cttcgaaggc ttaactctga aacatcacac atctaagatt 300 caagagtttg ccgacctaac tcaggttgaa acttttggct ttcgggggga agctctgagc 360 tcactttgtg cactgagcga tgtcaccatt tctacctgcc acgcatcggc gaaggttgga 420 actcgactga tgtttgatca caatgggaaa attatccaga aaacccccta cccccgcccc 480 agagggacca cagtcagcgt gcagcagtta ttttccacac tacctgtgcg ccataaggaa 540 tttcaaagga atattaagaa ggagtatgcc aaaatggtcc aggtcttaca tgcatactgt 600 atcatttcag caggcatccg tgtaagttgc accaatcagc ttggacaagg aaaacgacag 660 cctgtggtat gcacaggtgg aagccccagc ataaaggaaa atatcggctc tgtgtttggg 720 cagaagcagt tgcaaagcct cattcctttt gttcagctgc cccctagtga ctccgtgtgt 780 gaagagtacg gtttgagctg ttcggatgct ctgcataatc ttttttacat ctcaggtttc 840 atttcacaat gcacgcatgg agttggaagg agttcaacag acagacagtt tttctttatc 900 aaccggcggc cttgtgaccc agcaaaggtc tgcagactcg tgaatgaggt ctaccacatg 960 tataatcgac accagtatcc atttgttgtt cttaacattt ctgttgattc agaatgcgtt 1020 gatatcaatg ttactccaga taaaaggcaa attttgctac aagaggaaaa gcttttgttg 1080 gcagttttaa agacctcttt gataggaatg tttgatagtg atgtcaacaa gctaaatgtc 1140 agtcagcagc cactgctgga tgttgaaggt aacttaataa aaatgcatgc agcggatttg 1200 gaaaagccca tggtagaaaa gcaggatcaa tccccttcat taaggactgg agaagaaaaa 1260 aaagacgtgt ccatttccag actgcgagag gccttttctc ttcgtcacac aacagagaac 1320 aagcctcaca gcccaaagac tccagaacca agaaggagcc ctctaggaca gaaaaggggt 1380 atgctgtctt ctagcacttc aggtgccatc tctgacaaag gcgtcctgag acctcagaaa 1440 gaggcagtga gttccagtca cggacccagt gaccctacgg acagagcgga ggtggagaag 1500 gactcggggc acggcagcac ttccgtggat tctgaggggt tcagcatccc agacacgggc 1560 agtcactgca gcagcgagta tgcggccagc tccccagggg acaggggctc gcaggaacat 1620 gtggactctc aggagaaagc gcctgaaact gacgactctt tttcagatgt ggactgccat 1680 tcaaaccagg aagataccgg atgtaaattt cgagttttgc ctcagccaac taatctcgca 1740 accccaaaca caaagcgttt taaaaaagaa gaaattcttt ccagttctga catttgtcaa 1800 aagttagtaa atactcagga catgtcagcc tctcaggttg atgtagctgt gaaaattaat 1860 aagaaagttg tgcccctgga cttttctatg agttctttag ctaaacgaat aaagcagtta 1920 catcatgaag cacagcaaag tgaaggggaa cagaattaca ggaagtttag ggcaaagatt 1980 tgtcctggag aaaatcaagc agccgaagat gaactaagaa aagagataag taaaacgatg 2040 tttgcagaaa tggaaatcat tggtcagttt aacctgggat ttataataac caaactgaat 2100 gaggatatct tcatagtgga ccagcatgcc acggacgaga agtataactt cgagatgctg 2160 cagcagcaca ccgtgctcca ggggcagagg ctcatagcac ctcagactct caacttaact 2220 gctgttaatg aagctgttct gatagaaaat ctggaaatat ttagaaagaa tggctttgat 2280 tttgttatcg atgaaaatgc tccagtcact gaaagggcta aactgatttc cttgccaact 2340 agtaaaaact ggaccttcgg accccaggac gtcgatgaac tgatcttcat gctgagcgac 2400 agccctgggg tcatgtgccg gccttcccga gtcaagcaga tgtttgcctc cagagcctgc 2460 cggaagtcgg tgatgattgg gactgctctt aacacaagcg agatgaagaa actgatcacc 2520 cacatggggg agatggacca cccctggaac tgtccccatg gaaggccaac catgagacac 2580 atcgccaacc tgggtgtcat ttctcagaac tgaccgtagt cactgtatgg aataattggt 2640 tttatcgcag atttttatgt tttgaaagac agagtcttca ctaacctttt ttgttttaaa 2700 atgaaacctg ctacttaaaa aaaatacaca tcacacccat ttaaaagtga tcttgagaac 2760 cttttcaaac c 2771 9 932 PRT Homo sapiens 9 Met Lys Gln Leu Pro Ala Ala Thr Val Arg Leu Leu Ser Ser Ser Gln 1 5 10 15 Ile Ile Thr Ser Val Val Ser Val Val Lys Glu Leu Ile Glu Asn Ser 20 25 30 Leu Asp Ala Gly Ala Thr Ser Val Asp Val Lys Leu Glu Asn Tyr Gly 35 40 45 Phe Asp Lys Ile Glu Val Arg Asp Asn Gly Glu Gly Ile Lys Ala Val 50 55 60 Asp Ala Pro Val Met Ala Met Lys Tyr Tyr Thr Ser Lys Ile Asn Ser 65 70 75 80 His Glu Asp Leu Glu Asn Leu Thr Thr Tyr Gly Phe Arg Gly Glu Ala 85 90 95 Leu Gly Ser Ile Cys Cys Ile Ala Glu Val Leu Ile Thr Thr Arg Thr 100 105 110 Ala Ala Asp Asn Phe Ser Thr Gln Tyr Val Leu Asp Gly Ser Gly His 115 120 125 Ile Leu Ser Gln Lys Pro Ser His Leu Gly Gln Gly Thr Thr Val Thr 130 135 140 Ala Leu Arg Leu Phe Lys Asn Leu Pro Val Arg Lys Gln Phe Tyr Ser 145 150 155 160 Thr Ala Lys Lys Cys Lys Asp Glu Ile Lys Lys Ile Gln Asp Leu Leu 165 170 175 Met Ser Phe Gly Ile Leu Lys Pro Asp Leu Arg Ile Val Phe Val His 180 185 190 Asn Lys Ala Val Ile Trp Gln Lys Ser Arg Val Ser Asp His Lys Met 195 200 205 Ala Leu Met Ser Val Leu Gly Thr Ala Val Met Asn Asn Met Glu Ser 210 215 220 Phe Gln Tyr His Ser Glu Glu Ser Gln Ile Tyr Leu Ser Gly Phe Leu 225 230 235 240 Pro Lys Cys Asp Ala Asp His Ser Phe Thr Ser Leu Ser Thr Pro Glu 245 250 255 Arg Ser Phe Ile Phe Ile Asn Ser Arg Pro Val His Gln Lys Asp Ile 260 265 270 Leu Lys Leu Ile Arg His His Tyr Asn Leu Lys Cys Leu Lys Glu Ser 275 280 285 Thr Arg Leu Tyr Pro Val Phe Phe Leu Lys Ile Asp Val Pro Thr Ala 290 295 300 Asp Val Asp Val Asn Leu Thr Pro Asp Lys Ser Gln Val Leu Leu Gln 305 310 315 320 Asn Lys Glu Ser Val Leu Ile Ala Leu Glu Asn Leu Met Thr Thr Cys 325 330 335 Tyr Gly Pro Leu Pro Ser Thr Asn Ser Tyr Glu Asn Asn Lys Thr Asp 340 345 350 Val Ser Ala Ala Asp Ile Val Leu Ser Lys Thr Ala Glu Thr Asp Val 355 360 365 Leu Phe Asn Lys Val Glu Ser Ser Gly Lys Asn Tyr Ser Asn Val Asp 370 375 380 Thr Ser Val Ile Pro Phe Gln Asn Asp Met His Asn Asp Glu Ser Gly 385 390 395 400 Lys Asn Thr Asp Asp Cys Leu Asn His Gln Ile Ser Ile Gly Asp Phe 405 410 415 Gly Tyr Gly His Cys Ser Ser Glu Ile Ser Asn Ile Asp Lys Asn Thr 420 425 430 Lys Asn Ala Phe Gln Asp Ile Ser Met Ser Asn Val Ser Trp Glu Asn 435 440 445 Ser Gln Thr Glu Tyr Ser Lys Thr Cys Phe Ile Ser Ser Val Lys His 450 455 460 Thr Gln Ser Glu Asn Gly Asn Lys Asp His Ile Asp Glu Ser Gly Glu 465 470 475 480 Asn Glu Glu Glu Ala Gly Leu Glu Asn Ser Ser Glu Ile Ser Ala Asp 485 490 495 Glu Trp Ser Arg Gly Asn Ile Leu Lys Asn Ser Val Gly Glu Asn Ile 500 505 510 Glu Pro Val Lys Ile Leu Val Pro Glu Lys Ser Leu Pro Cys Lys Val 515 520 525 Ser Asn Asn Asn Tyr Pro Ile Pro Glu Gln Met Asn Leu Asn Glu Asp 530 535 540 Ser Cys Asn Lys Lys Ser Asn Val Ile Asp Asn Lys Ser Gly Lys Val 545 550 555 560 Thr Ala Tyr Asp Leu Leu Ser Asn Arg Val Ile Lys Lys Pro Met Ser 565 570 575 Ala Ser Ala Leu Phe Val Gln Asp His Arg Pro Gln Phe Leu Ile Glu 580 585 590 Asn Pro Lys Thr Ser Leu Glu Asp Ala Thr Leu Gln Ile Glu Glu Leu 595 600 605 Trp Lys Thr Leu Ser Glu Glu Glu Lys Leu Lys Tyr Glu Glu Lys Ala 610 615 620 Thr Lys Asp Leu Glu Arg Tyr Asn Ser Gln Met Lys Arg Ala Ile Glu 625 630 635 640 Gln Glu Ser Gln Met Ser Leu Lys Asp Gly Arg Lys Lys Ile Lys Pro 645 650 655 Thr Ser Ala Trp Asn Leu Ala Gln Lys His Lys Leu Lys Thr Ser Leu 660 665 670 Ser Asn Gln Pro Lys Leu Asp Glu Leu Leu Gln Ser Gln Ile Glu Lys 675 680 685 Arg Arg Ser Gln Asn Ile Lys Met Val Gln Ile Pro Phe Ser Met Lys 690 695 700 Asn Leu Lys Ile Asn Phe Lys Lys Gln Asn Lys Val Asp Leu Glu Glu 705 710 715 720 Lys Asp Glu Pro Cys Leu Ile His Asn Leu Arg Phe Pro Asp Ala Trp 725 730 735 Leu Met Thr Ser Lys Thr Glu Val Met Leu Leu Asn Pro Tyr Arg Val 740 745 750 Glu Glu Ala Leu Leu Phe Lys Arg Leu Leu Glu Asn His Lys Leu Pro 755 760 765 Ala Glu Pro Leu Glu Lys Pro Ile Met Leu Thr Glu Ser Leu Phe Asn 770 775 780 Gly Ser His Tyr Leu Asp Val Leu Tyr Lys Met Thr Ala Asp Asp Gln 785 790 795 800 Arg Tyr Ser Gly Ser Thr Tyr Leu Ser Asp Pro Arg Leu Thr Ala Asn 805 810 815 Gly Phe Lys Ile Lys Leu Ile Pro Gly Val Ser Ile Thr Glu Asn Tyr 820 825 830 Leu Glu Ile Glu Gly Met Ala Asn Cys Leu Pro Phe Tyr Gly Val Ala 835 840 845 Asp Leu Lys Glu Ile Leu Asn Ala Ile Leu Asn Arg Asn Ala Lys Glu 850 855 860 Val Tyr Glu Cys Arg Pro Arg Lys Val Ile Ser Tyr Leu Glu Gly Glu 865 870 875 880 Ala Val Arg Leu Ser Arg Gln Leu Pro Met Tyr Leu Ser Lys Glu Asp 885 890 895 Ile Gln Asp Ile Ile Tyr Arg Met Lys His Gln Phe Gly Asn Glu Ile 900 905 910 Lys Glu Cys Val His Gly Arg Pro Phe Phe His His Leu Thr Tyr Leu 915 920 925 Pro Glu Thr Thr 930 10 3063 DNA Homo sapiens 10 ggcacgagtg gctgcttgcg gctagtggat ggtaattgcc tgcctcgcgc tagcagcaag 60 ctgctctgtt aaaagcgaaa atgaaacaat tgcctgcggc aacagttcga ctcctttcaa 120 gttctcagat catcacttcg gtggtcagtg ttgtaaaaga gcttattgaa aactccttgg 180 atgctggtgc cacaagcgta gatgttaaac tggagaacta tggatttgat aaaattgagg 240 tgcgagataa cggggagggt atcaaggctg ttgatgcacc tgtaatggca atgaagtact 300 acacctcaaa aataaatagt catgaagatc ttgaaaattt gacaacttac ggttttcgtg 360 gagaagcctt ggggtcaatt tgttgtatag ctgaggtttt aattacaaca agaacggctg 420 ctgataattt tagcacccag tatgttttag atggcagtgg ccacatactt tctcagaaac 480 cttcacatct tggtcaaggt acaactgtaa ctgctttaag attatttaag aatctacctg 540 taagaaagca gttttactca actgcaaaaa aatgtaaaga tgaaataaaa aagatccaag 600 atctcctcat gagctttggt atccttaaac ctgacttaag gattgtcttt gtacataaca 660 aggcagttat ttggcagaaa agcagagtat cagatcacaa gatggctctc atgtcagttc 720 tggggactgc tgttatgaac aatatggaat cctttcagta ccactctgaa gaatctcaga 780 tttatctcag tggatttctt ccaaagtgtg atgcagacca ctctttcact agtctttcaa 840 caccagaaag aagtttcatc ttcataaaca gtcgaccagt acatcaaaaa gatatcttaa 900 agttaatccg acatcattac aatctgaaat gcctaaagga atctactcgt ttgtatcctg 960 ttttctttct gaaaatcgat gttcctacag ctgatgttga tgtaaattta acaccagata 1020 aaagccaagt attattacaa aataaggaat ctgttttaat tgctcttgaa aatctgatga 1080 cgacttgtta tggaccatta cctagtacaa attcttatga aaataataaa acagatgttt 1140 ccgcagctga catcgttctt agtaaaacag cagaaacaga tgtgcttttt aataaagtgg 1200 aatcatctgg aaagaattat tcaaatgttg atacttcagt cattccattc caaaatgata 1260 tgcataatga tgaatctgga aaaaacactg atgattgttt aaatcaccag ataagtattg 1320 gtgactttgg ttatggtcat tgtagtagtg aaatttctaa cattgataaa aacactaaga 1380 atgcatttca ggacatttca atgagtaatg tatcatggga gaactctcag acggaatata 1440 gtaaaacttg ttttataagt tccgttaagc acacccagtc agaaaatggc aataaagacc 1500 atatagatga gagtggggaa aatgaggaag aagcaggtct tgaaaactct tcggaaattt 1560 ctgcagatga gtggagcagg ggaaatatac ttaaaaattc agtgggagag aatattgaac 1620 ctgtgaaaat tttagtgcct gaaaaaagtt taccatgtaa agtaagtaat aataattatc 1680 caatccctga acaaatgaat cttaatgaag attcatgtaa caaaaaatca aatgtaatag 1740 ataataaatc tggaaaagtt acagcttatg atttacttag caatcgagta atcaagaaac 1800 ccatgtcagc aagtgctctt tttgttcaag atcatcgtcc tcagtttctc atagaaaatc 1860 ctaagactag tttagaggat gcaacactac aaattgaaga actgtggaag acattgagtg 1920 aagaggaaaa actgaaatat gaagagaagg ctactaaaga cttggaacga tacaatagtc 1980 aaatgaagag agccattgaa caggagtcac aaatgtcact aaaagatggc agaaaaaaga 2040 taaaacccac cagcgcatgg aatttggccc agaagcacaa gttaaaaacc tcattatcta 2100 atcaaccaaa acttgatgaa ctccttcagt cccaaattga aaaaagaagg agtcaaaata 2160 ttaaaatggt acagatcccc ttttctatga aaaacttaaa aataaatttt aagaaacaaa 2220 acaaagttga cttagaagag aaggatgaac cttgcttgat ccacaatctc aggtttcctg 2280 atgcatggct aatgacatcc aaaacagagg taatgttatt aaatccatat agagtagaag 2340 aagccctgct atttaaaaga cttcttgaga atcataaact tcctgcagag ccactggaaa 2400 agccaattat gttaacagag agtcttttta atggatctca ttatttagac gttttatata 2460 aaatgacagc agatgaccaa agatacagtg gatcaactta cctgtctgat cctcgtctta 2520 cagcgaatgg tttcaagata aaattgatac caggagtttc aattactgaa aattacttgg 2580 aaatagaagg aatggctaat tgtctcccat tctatggagt agcagattta aaagaaattc 2640 ttaatgctat attaaacaga aatgcaaagg aagtttatga atgtagacct cgcaaagtga 2700 taagttattt agagggagaa gcagtgcgtc tatccagaca attacccatg tacttatcaa 2760 aagaggacat ccaagacatt atctacagaa tgaagcacca gtttggaaat gaaattaaag 2820 agtgtgttca tggtcgccca ttttttcatc atttaaccta tcttccagaa actacatgat 2880 taaatatgtt taagaagatt agttaccatt gaaattggtt ctgtcataaa acagcatgag 2940 tctggtttta aattatcttt gtattatgtg tcacatggtt attttttaaa tgaggattca 3000 ctgacttgtt tttatattga aaaaagttcc acgtattgta gaaaacgtaa ataaactaat 3060 aac 3063 11 934 PRT Homo sapiens 11 Met Ala Val Gln Pro Lys Glu Thr Leu Gln Leu Glu Ser Ala Ala Glu 1 5 10 15 Val Gly Phe Val Arg Phe Phe Gln Gly Met Pro Glu Lys Pro Thr Thr 20 25 30 Thr Val Arg Leu Phe Asp Arg Gly Asp Phe Tyr Thr Ala His Gly Glu 35 40 45 Asp Ala Leu Leu Ala Ala Arg Glu Val Phe Lys Thr Gln Gly Val Ile 50 55 60 Lys Tyr Met Gly Pro Ala Gly Ala Lys Asn Leu Gln Ser Val Val Leu 65 70 75 80 Ser Lys Met Asn Phe Glu Ser Phe Val Lys Asp Leu Leu Leu Val Arg 85 90 95 Gln Tyr Arg Val Glu Val Tyr Lys Asn Arg Ala Gly Asn Lys Ala Ser 100 105 110 Lys Glu Asn Asp Trp Tyr Leu Ala Tyr Lys Ala Ser Pro Gly Asn Leu 115 120 125 Ser Gln Phe Glu Asp Ile Leu Phe Gly Asn Asn Asp Met Ser Ala Ser 130 135 140 Ile Gly Val Val Gly Val Lys Met Ser Ala Val Asp Gly Gln Arg Gln 145 150 155 160 Val Gly Val Gly Tyr Val Asp Ser Ile Gln Arg Lys Leu Gly Leu Cys 165 170 175 Glu Phe Pro Asp Asn Asp Gln Phe Ser Asn Leu Glu Ala Leu Leu Ile 180 185 190 Gln Ile Gly Pro Lys Glu Cys Val Leu Pro Gly Gly Glu Thr Ala Gly 195 200 205 Asp Met Gly Lys Leu Arg Gln Ile Ile Gln Arg Gly Gly Ile Leu Ile 210 215 220 Thr Glu Arg Lys Lys Ala Asp Phe Ser Thr Lys Asp Ile Tyr Gln Asp 225 230 235 240 Leu Asn Arg Leu Leu Lys Gly Lys Lys Gly Glu Gln Met Asn Ser Ala 245 250 255 Val Leu Pro Glu Met Glu Asn Gln Val Ala Val Ser Ser Leu Ser Ala 260 265 270 Val Ile Lys Phe Leu Glu Leu Leu Ser Asp Asp Ser Asn Phe Gly Gln 275 280 285 Phe Glu Leu Thr Thr Phe Asp Phe Ser Gln Tyr Met Lys Leu Asp Ile 290 295 300 Ala Ala Val Arg Ala Leu Asn Leu Phe Gln Gly Ser Val Glu Asp Thr 305 310 315 320 Thr Gly Ser Gln Ser Leu Ala Ala Leu Leu Asn Lys Cys Lys Thr Pro 325 330 335 Gln Gly Gln Arg Leu Val Asn Gln Trp Ile Lys Gln Pro Leu Met Asp 340 345 350 Lys Asn Arg Ile Glu Glu Arg Leu Asn Leu Val Glu Ala Phe Val Glu 355 360 365 Asp Ala Glu Leu Arg Gln Thr Leu Gln Glu Asp Leu Leu Arg Arg Phe 370 375 380 Pro Asp Leu Asn Arg Leu Ala Lys Lys Phe Gln Arg Gln Ala Ala Asn 385 390 395 400 Leu Gln Asp Cys Tyr Arg Leu Tyr Gln Gly Ile Asn Gln Leu Pro Asn 405 410 415 Val Ile Gln Ala Leu Glu Lys His Glu Gly Lys His Gln Lys Leu Leu 420 425 430 Leu Ala Val Phe Val Thr Pro Leu Thr Asp Leu Arg Ser Asp Phe Ser 435 440 445 Lys Phe Gln Glu Met Ile Glu Thr Thr Leu Asp Met Asp Gln Val Glu 450 455 460 Asn His Glu Phe Leu Val Lys Pro Ser Phe Asp Pro Asn Leu Ser Glu 465 470 475 480 Leu Arg Glu Ile Met Asn Asp Leu Glu Lys Lys Met Gln Ser Thr Leu 485 490 495 Ile Ser Ala Ala Arg Asp Leu Gly Leu Asp Pro Gly Lys Gln Ile Lys 500 505 510 Leu Asp Ser Ser Ala Gln Phe Gly Tyr Tyr Phe Arg Val Thr Cys Lys 515 520 525 Glu Glu Lys Val Leu Arg Asn Asn Lys Asn Phe Ser Thr Val Asp Ile 530 535 540 Gln Lys Asn Gly Val Lys Phe Thr Asn Ser Lys Leu Thr Ser Leu Asn 545 550 555 560 Glu Glu Tyr Thr Lys Asn Lys Thr Glu Tyr Glu Glu Ala Gln Asp Ala 565 570 575 Ile Val Lys Glu Ile Val Asn Ile Ser Ser Gly Tyr Val Glu Pro Met 580 585 590 Gln Thr Leu Asn Asp Val Leu Ala Gln Leu Asp Ala Val Val Ser Phe 595 600 605 Ala His Val Ser Asn Gly Ala Pro Val Pro Tyr Val Arg Pro Ala Ile 610 615 620 Leu Glu Lys Gly Gln Gly Arg Ile Ile Leu Lys Ala Ser Arg His Ala 625 630 635 640 Cys Val Glu Val Gln Asp Glu Ile Ala Phe Ile Pro Asn Asp Val Tyr 645 650 655 Phe Glu Lys Asp Lys Gln Met Phe His Ile Ile Thr Gly Pro Asn Met 660 665 670 Gly Gly Lys Ser Thr Tyr Ile Arg Gln Thr Gly Val Ile Val Leu Met 675 680 685 Ala Gln Ile Gly Cys Phe Val Pro Cys Glu Ser Ala Glu Val Ser Ile 690 695 700 Val Asp Cys Ile Leu Ala Arg Val Gly Ala Gly Asp Ser Gln Leu Lys 705 710 715 720 Gly Val Ser Thr Phe Met Ala Glu Met Leu Glu Thr Ala Ser Ile Leu 725 730 735 Arg Ser Ala Thr Lys Asp Ser Leu Ile Ile Ile Asp Glu Leu Gly Arg 740 745 750 Gly Thr Ser Thr Tyr Asp Gly Phe Gly Leu Ala Trp Ala Ile Ser Glu 755 760 765 Tyr Ile Ala Thr Lys Ile Gly Ala Phe Cys Met Phe Ala Thr His Phe 770 775 780 His Glu Leu Thr Ala Leu Ala Asn Gln Ile Pro Thr Val Asn Asn Leu 785 790 795 800 His Val Thr Ala Leu Thr Thr Glu Glu Thr Leu Thr Met Leu Tyr Gln 805 810 815 Val Lys Lys Gly Val Cys Asp Gln Ser Phe Gly Ile His Val Ala Glu 820 825 830 Leu Ala Asn Phe Pro Lys His Val Ile Glu Cys Ala Lys Gln Lys Ala 835 840 845 Leu Glu Leu Glu Glu Phe Gln Tyr Ile Gly Glu Ser Gln Gly Tyr Asp 850 855 860 Ile Met Glu Pro Ala Ala Lys Lys Cys Tyr Leu Glu Arg Glu Gln Gly 865 870 875 880 Glu Lys Ile Ile Gln Glu Phe Leu Ser Lys Val Lys Gln Met Pro Phe 885 890 895 Thr Glu Met Ser Glu Glu Asn Ile Thr Ile Lys Leu Lys Gln Leu Lys 900 905 910 Ala Glu Val Ile Ala Lys Asn Asn Ser Phe Val Asn Glu Ile Ile Ser 915 920 925 Arg Ile Lys Val Thr Thr 930 12 3145 DNA Homo sapiens 12 ggcgggaaac agcttagtgg gtgtggggtc gcgcattttc ttcaaccagg aggtgaggag 60 gtttcgacat ggcggtgcag ccgaaggaga cgctgcagtt ggagagcgcg gccgaggtcg 120 gcttcgtgcg cttctttcag ggcatgccgg agaagccgac caccacagtg cgccttttcg 180 accggggcga cttctatacg gcgcacggcg aggacgcgct gctggccgcc cgggaggtgt 240 tcaagaccca gggggtgatc aagtacatgg ggccggcagg agcaaagaat ctgcagagtg 300 ttgtgcttag taaaatgaat tttgaatctt ttgtaaaaga tcttcttctg gttcgtcagt 360 atagagttga agtttataag aatagagctg gaaataaggc atccaaggag aatgattggt 420 atttggcata taaggcttct cctggcaatc tctctcagtt tgaagacatt ctctttggta 480 acaatgatat gtcagcttcc attggtgttg tgggtgttaa aatgtccgca gttgatggcc 540 agagacaggt tggagttggg tatgtggatt ccatacagag gaaactagga ctgtgtgaat 600 tccctgataa tgatcagttc tccaatcttg aggctctcct catccagatt ggaccaaagg 660 aatgtgtttt acccggagga gagactgctg gagacatggg gaaactgaga cagataattc 720 aaagaggagg aattctgatc acagaaagaa aaaaagctga cttttccaca aaagacattt 780 atcaggacct caaccggttg ttgaaaggca aaaagggaga gcagatgaat agtgctgtat 840 tgccagaaat ggagaatcag gttgcagttt catcactgtc tgcggtaatc aagtttttag 900 aactcttatc agatgattcc aactttggac agtttgaact gactactttt gacttcagcc 960 agtatatgaa attggatatt gcagcagtca gagcccttaa cctttttcag ggttctgttg 1020 aagataccac tggctctcag tctctggctg ccttgctgaa taagtgtaaa acccctcaag 1080 gacaaagact tgttaaccag tggattaagc agcctctcat ggataagaac agaatagagg 1140 agagattgaa tttagtggaa gcttttgtag aagatgcaga attgaggcag actttacaag 1200 aagatttact tcgtcgattc ccagatctta accgacttgc caagaagttt caaagacaag 1260 cagcaaactt acaagattgt taccgactct atcagggtat aaatcaacta cctaatgtta 1320 tacaggctct ggaaaaacat gaaggaaaac accagaaatt attgttggca gtttttgtga 1380 ctcctcttac tgatcttcgt tctgacttct ccaagtttca ggaaatgata gaaacaactt 1440 tagatatgga tcaggtggaa aaccatgaat tccttgtaaa accttcattt gatcctaatc 1500 tcagtgaatt aagagaaata atgaatgact tggaaaagaa gatgcagtca acattaataa 1560 gtgcagccag agatcttggc ttggaccctg gcaaacagat taaactggat tccagtgcac 1620 agtttggata ttactttcgt gtaacctgta aggaagaaaa agtccttcgt aacaataaaa 1680 actttagtac tgtagatatc cagaagaatg gtgttaaatt taccaacagc aaattgactt 1740 ctttaaatga agagtatacc aaaaataaaa cagaatatga agaagcccag gatgccattg 1800 ttaaagaaat tgtcaatatt tcttcaggct atgtagaacc aatgcagaca ctcaatgatg 1860 tgttagctca gctagatgct gttgtcagct ttgctcacgt gtcaaatgga gcacctgttc 1920 catatgtacg accagccatt ttggagaaag gacaaggaag aattatatta aaagcatcca 1980 ggcatgcttg tgttgaagtt caagatgaaa ttgcatttat tcctaatgac gtatactttg 2040 aaaaagataa acagatgttc cacatcatta ctggccccaa tatgggaggt aaatcaacat 2100 atattcgaca aactggggtg atagtactca tggcccaaat tgggtgtttt gtgccatgtg 2160 agtcagcaga agtgtccatt gtggactgca tcttagcccg agtaggggct ggtgacagtc 2220 aattgaaagg agtctccacg ttcatggctg aaatgttgga aactgcttct atcctcaggt 2280 ctgcaaccaa agattcatta ataatcatag atgaattggg aagaggaact tctacctacg 2340 atggatttgg gttagcatgg gctatatcag aatacattgc aacaaagatt ggtgcttttt 2400 gcatgtttgc aacccatttt catgaactta ctgccttggc caatcagata ccaactgtta 2460 ataatctaca tgtcacagca ctcaccactg aagagacctt aactatgctt tatcaggtga 2520 agaaaggtgt ctgtgatcaa agttttggga ttcatgttgc agagcttgct aatttcccta 2580 agcatgtaat agagtgtgct aaacagaaag ccctggaact tgaggagttt cagtatattg 2640 gagaatcgca aggatatgat atcatggaac cagcagcaaa gaagtgctat ctggaaagag 2700 agcaaggtga aaaaattatt caggagttcc tgtccaaggt gaaacaaatg ccctttactg 2760 aaatgtcaga agaaaacatc acaataaagt taaaacagct aaaagctgaa gtaatagcaa 2820 agaataatag ctttgtaaat gaaatcattt cacgaataaa agttactacg tgaaaaatcc 2880 cagtaatgga atgaaggtaa tattgataag ctattgtctg taatagtttt atattgtttt 2940 atattaaccc tttttccata gtgttaactg tcagtgccca tgggctatca acttaataag 3000 atatttagta atattttact ttgaggacat tttcaaagat ttttattttg aaaaatgaga 3060 gctgtaactg aggactgttt gcaattgaca taggcaataa taagtgatgt gctgaatttt 3120 ataaataaaa tcatgtagtt tgtgg 3145 13 756 PRT Homo sapiens 13 Met Ser Phe Val Ala Gly Val Ile Arg Arg Leu Asp Glu Thr Val Val 1 5 10 15 Asn Arg Ile Ala Ala Gly Glu Val Ile Gln Arg Pro Ala Asn Ala Ile 20 25 30 Lys Glu Met Ile Glu Asn Cys Leu Asp Ala Lys Ser Thr Ser Ile Gln 35 40 45 Val Ile Val Lys Glu Gly Gly Leu Lys Leu Ile Gln Ile Gln Asp Asn 50 55 60 Gly Thr Gly Ile Arg Lys Glu Asp Leu Asp Ile Val Cys Glu Arg Phe 65 70 75 80 Thr Thr Ser Lys Leu Gln Ser Phe Glu Asp Leu Ala Ser Ile Ser Thr 85 90 95 Tyr Gly Phe Arg Gly Glu Ala Leu Ala Ser Ile Ser His Val Ala His 100 105 110 Val Thr Ile Thr Thr Lys Thr Ala Asp Gly Lys Cys Ala Tyr Arg Ala 115 120 125 Ser Tyr Ser Asp Gly Lys Leu Lys Ala Pro Pro Lys Pro Cys Ala Gly 130 135 140 Asn Gln Gly Thr Gln Ile Thr Val Glu Asp Leu Phe Tyr Asn Ile Ala 145 150 155 160 Thr Arg Arg Lys Ala Leu Lys Asn Pro Ser Glu Glu Tyr Gly Lys Ile 165 170 175 Leu Glu Val Val Gly Arg Tyr Ser Val His Asn Ala Gly Ile Ser Phe 180 185 190 Ser Val Lys Lys Gln Gly Glu Thr Val Ala Asp Val Arg Thr Leu Pro 195 200 205 Asn Ala Ser Thr Val Asp Asn Ile Arg Ser Ile Phe Gly Asn Ala Val 210 215 220 Ser Arg Glu Leu Ile Glu Ile Gly Cys Glu Asp Lys Thr Leu Ala Phe 225 230 235 240 Lys Met Asn Gly Tyr Ile Ser Asn Ala Asn Tyr Ser Val Lys Lys Cys 245 250 255 Ile Phe Leu Leu Phe Ile Asn His Arg Leu Val Glu Ser Thr Ser Leu 260 265 270 Arg Lys Ala Ile Glu Thr Val Tyr Ala Ala Tyr Leu Pro Lys Asn Thr 275 280 285 His Pro Phe Leu Tyr Leu Ser Leu Glu Ile Ser Pro Gln Asn Val Asp 290 295 300 Val Asn Val His Pro Thr Lys His Glu Val His Phe Leu His Glu Glu 305 310 315 320 Ser Ile Leu Glu Arg Val Gln Gln His Ile Glu Ser Lys Leu Leu Gly 325 330 335 Ser Asn Ser Ser Arg Met Tyr Phe Thr Gln Thr Leu Leu Pro Gly Leu 340 345 350 Ala Gly Pro Ser Gly Glu Met Val Lys Ser Thr Thr Ser Leu Thr Ser 355 360 365 Ser Ser Thr Ser Gly Ser Ser Asp Lys Val Tyr Ala His Gln Met Val 370 375 380 Arg Thr Asp Ser Arg Glu Gln Lys Leu Asp Ala Phe Leu Gln Pro Leu 385 390 395 400 Ser Lys Pro Leu Ser Ser Gln Pro Gln Ala Ile Val Thr Glu Asp Lys 405 410 415 Thr Asp Ile Ser Ser Gly Arg Ala Arg Gln Gln Asp Glu Glu Met Leu 420 425 430 Glu Leu Pro Ala Pro Ala Glu Val Ala Ala Lys Asn Gln Ser Leu Glu 435 440 445 Gly Asp Thr Thr Lys Gly Thr Ser Glu Met Ser Glu Lys Arg Gly Pro 450 455 460 Thr Ser Ser Asn Pro Arg Lys Arg His Arg Glu Asp Ser Asp Val Glu 465 470 475 480 Met Val Glu Asp Asp Ser Arg Lys Glu Met Thr Ala Ala Cys Thr Pro 485 490 495 Arg Arg Arg Ile Ile Asn Leu Thr Ser Val Leu Ser Leu Gln Glu Glu 500 505 510 Ile Asn Glu Gln Gly His Glu Val Leu Arg Glu Met Leu His Asn His 515 520 525 Ser Phe Val Gly Cys Val Asn Pro Gln Trp Ala Leu Ala Gln His Gln 530 535 540 Thr Lys Leu Tyr Leu Leu Asn Thr Thr Lys Leu Ser Glu Glu Leu Phe 545 550 555 560 Tyr Gln Ile Leu Ile Tyr Asp Phe Ala Asn Phe Gly Val Leu Arg Leu 565 570 575 Ser Glu Pro Ala Pro Leu Phe Asp Leu Ala Met Leu Ala Leu Asp Ser 580 585 590 Pro Glu Ser Gly Trp Thr Glu Glu Asp Gly Pro Lys Glu Gly Leu Ala 595 600 605 Glu Tyr Ile Val Glu Phe Leu Lys Lys Lys Ala Glu Met Leu Ala Asp 610 615 620 Tyr Phe Ser Leu Glu Ile Asp Glu Glu Gly Asn Leu Ile Gly Leu Pro 625 630 635 640 Leu Leu Ile Asp Asn Tyr Val Pro Pro Leu Glu Gly Leu Pro Ile Phe 645 650 655 Ile Leu Arg Leu Ala Thr Glu Val Asn Trp Asp Glu Glu Lys Glu Cys 660 665 670 Phe Glu Ser Leu Ser Lys Glu Cys Ala Met Phe Tyr Ser Ile Arg Lys 675 680 685 Gln Tyr Ile Ser Glu Glu Ser Thr Leu Ser Gly Gln Gln Ser Glu Val 690 695 700 Pro Gly Ser Ile Pro Asn Ser Trp Lys Trp Thr Val Glu His Ile Val 705 710 715 720 Tyr Lys Ala Leu Arg Ser His Ile Leu Pro Pro Lys His Phe Thr Glu 725 730 735 Asp Gly Asn Ile Leu Gln Leu Ala Asn Leu Pro Asp Leu Tyr Lys Val 740 745 750 Phe Glu Arg Cys 755 14 2484 DNA Homo sapiens 14 cttggctctt ctggcgccaa aatgtcgttc gtggcagggg ttattcggcg gctggacgag 60 acagtggtga accgcatcgc ggcgggggaa gttatccagc ggccagctaa tgctatcaaa 120 gagatgattg agaactgttt agatgcaaaa tccacaagta ttcaagtgat tgttaaagag 180 ggaggcctga agttgattca gatccaagac aatggcaccg ggatcaggaa agaagatctg 240 gatattgtat gtgaaaggtt cactactagt aaactgcagt cctttgagga tttagccagt 300 atttctacct atggctttcg aggtgaggct ttggccagca taagccatgt ggctcatgtt 360 actattacaa cgaaaacagc tgatggaaag tgtgcataca gagcaagtta ctcagatgga 420 aaactgaaag cccctcctaa accatgtgct ggcaatcaag ggacccagat cacggtggag 480 gacctttttt acaacatagc cacgaggaga aaagctttaa aaaatccaag tgaagaatat 540 gggaaaattt tggaagttgt tggcaggtat tcagtacaca atgcaggcat tagtttctca 600 gttaaaaaac aaggagagac agtagctgat gttaggacac tacccaatgc ctcaaccgtg 660 gacaatattc gctccatctt tggaaatgct gttagtcgag aactgataga aattggatgt 720 gaggataaaa ccctagcctt caaaatgaat ggttacatat ccaatgcaaa ctactcagtg 780 aagaagtgca tcttcttact cttcatcaac catcgtctgg tagaatcaac ttccttgaga 840 aaagccatag aaacagtgta tgcagcctat ttgcccaaaa acacacaccc attcctgtac 900 ctcagtttag aaatcagtcc ccagaatgtg gatgttaatg tgcaccccac aaagcatgaa 960 gttcacttcc tgcacgagga gagcatcctg gagcgggtgc agcagcacat cgagagcaag 1020 ctcctgggct ccaattcctc caggatgtac ttcacccaga ctttgctacc aggacttgct 1080 ggcccctctg gggagatggt taaatccaca acaagtctga cctcgtcttc tacttctgga 1140 agtagtgata aggtctatgc ccaccagatg gttcgtacag attcccggga acagaagctt 1200 gatgcatttc tgcagcctct gagcaaaccc ctgtccagtc agccccaggc cattgtcaca 1260 gaggataaga cagatatttc tagtggcagg gctaggcagc aagatgagga gatgcttgaa 1320 ctcccagccc ctgctgaagt ggctgccaaa aatcagagct tggaggggga tacaacaaag 1380 gggacttcag aaatgtcaga gaagagagga cctacttcca gcaaccccag aaagagacat 1440 cgggaagatt ctgatgtgga aatggtggaa gatgattccc gaaaggaaat gactgcagct 1500 tgtacccccc ggagaaggat cattaacctc actagtgttt tgagtctcca ggaagaaatt 1560 aatgagcagg gacatgaggt tctccgggag atgttgcata accactcctt cgtgggctgt 1620 gtgaatcctc agtgggcctt ggcacagcat caaaccaagt tataccttct caacaccacc 1680 aagcttagtg aagaactgtt ctaccagata ctcatttatg attttgccaa ttttggtgtt 1740 ctcaggttat cggagccagc accgctcttt gaccttgcca tgcttgcctt agatagtcca 1800 gagagtggct ggacagagga agatggtccc aaagaaggac ttgctgaata cattgttgag 1860 tttctgaaga agaaggctga gatgcttgca gactatttct ctttggaaat tgatgaggaa 1920 gggaacctga ttggattacc ccttctgatt gacaactatg tgcccccttt ggagggactg 1980 cctatcttca ttcttcgact agccactgag gtgaattggg acgaagaaaa ggaatgtttt 2040 gaaagcctca gtaaagaatg cgctatgttc tattccatcc ggaagcagta catatctgag 2100 gagtcgaccc tctcaggcca gcagagtgaa gtgcctggct ccattccaaa ctcctggaag 2160 tggactgtgg aacacattgt ctataaagcc ttgcgctcac acattctgcc tcctaaacat 2220 ttcacagaag atggaaatat cctgcagctt gctaacctgc ctgatctata caaagtcttt 2280 gagaggtgtt aaatatggtt atttatgcac tgtgggatgt gttcttcttt ctctgtattc 2340 cgatacaaag tgttgtatca aagtgtgata tacaaagtgt accaacataa gtgttggtag 2400 cacttaagac ttatacttgc cttctgatag tattccttta tacacagtgg attgattata 2460 aataaataga tgtgtcttaa cata 2484 15 133 PRT Homo sapiens 15 Met Glu Arg Ala Glu Ser Ser Ser Thr Glu Pro Ala Lys Ala Ile Lys 1 5 10 15 Pro Ile Asp Arg Lys Ser Val His Gln Ile Cys Ser Gly Gln Val Val 20 25 30 Leu Ser Leu Ser Thr Ala Val Lys Glu Leu Val Glu Asn Ser Leu Asp 35 40 45 Ala Gly Ala Thr Asn Ile Asp Leu Lys Leu Lys Asp Tyr Gly Val Asp 50 55 60 Leu Ile Glu Val Ser Asp Asn Gly Cys Gly Val Glu Glu Glu Asn Phe 65 70 75 80 Glu Gly Leu Thr Leu Lys His His Thr Ser Lys Ile Gln Glu Phe Ala 85 90 95 Asp Leu Thr Gln Val Glu Thr Phe Gly Phe Arg Gly Glu Ala Leu Ser 100 105 110 Ser Leu Cys Ala Leu Ser Asp Val Thr Ile Ser Thr Cys His Ala Ser 115 120 125 Ala Lys Val Gly Thr 130 16 426 DNA Homo sapiens 16 cgaggcggat cgggtgttgc atccatggag cgagctgaga gctcgagtac agaacctgct 60 aaggccatca aacctattga tcggaagtca gtccatcaga tttgctctgg gcaggtggta 120 ctgagtctaa gcactgcggt aaaggagtta gtagaaaaca gtctggatgc tggtgccact 180 aatattgatc taaagcttaa ggactatgga gtggatctta ttgaagtttc agacaatgga 240 tgtggggtag aagaagaaaa cttcgaaggc ttaactctga aacatcacac atctaagatt 300 caagagtttg ccgacctaac tcaggttgaa acttttggct ttcgggggga agctctgagc 360 tcactttgtg cactgagcga tgtcaccatt tctacctgcc acgcatcggc gaaggttgga 420 acttga 426 17 181 DNA Artificial Sequence Sec-hist expression cassette 17 aagcttccat gtacaggatg caactcctgt cttgcattgc actaagtctt gcacttgtca 60 caaacagtgc acaaaagctg gagctccacc gcggtggcgg ccgctctaga actagtggat 120 cccccggggc tgcaggaatt cgatatcaag ccaccatcac catcaccact agtagaagct 180 t 181 

We claim:
 1. A method for making a hypermutated antigen, comprising introducing into a mammalian cell that expresses a preselected antigen a polynucleotide comprising a dominant negative allele of a mismatch repair gene.
 2. The method of claim 1 wherein the polynucleotide is introduced by transfection of a suspension of cells in vitro.
 3. The method of claim 1 wherein the mismatch repair gene is PMS2.
 4. The method of claim 1 wherein the mismatch repair gene is human PMS2.
 5. The method of claim 1 wherein the mismatch repair gene is MLH1.
 6. The method of claim 1 wherein the mismatch repair gene is PMS1.
 7. The method of claim 1 wherein the mismatch repair gene is MSH2.
 8. The method of claim 1 wherein the mismatch repair gene is MSH2.
 9. The method of claim 4 wherein the allele comprises a truncation mutation.
 10. The method of claim 4 wherein the allele comprises a truncation mutation at codon
 134. 11. The method of claim 10 wherein the truncation mutation is a thymidine at nucleotide 424 of wild-type PMS2.
 12. The method of claim 1 wherein the polynucleotide is introduced into a fertilized egg of an animal.
 13. The method of claim 12 wherein the fertilized egg is subsequently implanted into a pseudo-pregnant female whereby the fertilized egg develops into a mature transgenic animal.
 14. The method of claim 12 wherein the mismatch repair gene is PMS2.
 15. The method of claim 12 wherein the mismatch repair gene is human PMS2.
 16. The method of claim 12 wherein the mismatch repair gene is human MLH1.
 17. The method of claim 12 wherein the mismatch repair gene is human PMS1.
 18. The method of claim 11 wherein the mismatch repair gene is human a human mutL homolog.
 19. The method of claim 15 wherein the allele comprises a truncation mutation.
 20. The method of claim 15 wherein the allele comprises a truncation mutation at codon
 134. 21. The method of claim 19 wherein the truncation mutation is a thymidine at nucleotide 424 of wild-type PMS2.
 22. A homogeneous composition of cultured, hypermutable, mammalian cells which comprise a preselected antigen and a dominant negative allele of a mismatch repair gene.
 23. The isolated hypermutable cell of claim 22 wherein the mismatch repair gene is PMS2.
 24. The isolated hypermutable cell of claim 23 wherein the mismatch repair gene is human PMS2.
 25. The isolated hypermutable cell of claim 22 wherein the mismatch repair gene is MLH1.
 26. The isolated hypermutable cell of claim 22 wherein the mismatch repair gene is PMS1.
 27. The isolated hypermutable cell of claim 22 wherein the mismatch repair gene is a human mutL homolog.
 28. The isolated hypermutable cell of claim 22 wherein the cells express a protein consisting of the first 133 amino acids of hPMS2.
 29. A method for generating a mutation in a gene encoding an antigen of interest comprising growing a mammalian cell comprising said gene encoding an antigen of interest and a dominant negative allele of a mismatch repair gene, and determining whether said gene encoding an antigen of interest harbors a mutation.
 30. The method of claim 29 wherein determining whether said gene encoding an antigen of interest harbors a mutation is accomplished by analyzing the nucleotide sequence of said gene.
 31. The method of claim 30 wherein said nucleotide sequence is an mRNA transcribed from said gene.
 32. The method of claim 29 wherein determining whether said gene encoding an antigen of interest harbors a mutation is accomplished by analyzing a protein encoded by said gene.
 33. The method of claim 30 wherein determining whether said gene encoding an antigen of interest harbors a mutation is accomplished by analyzing the phenotype of said gene.
 34. The method of claim 32 wherein analyzing of said protein comprises analyzing the antigenicity and immunogenicity of said protein.
 35. A method for generating a mutation in a gene encoding an antigen of interest comprising growing a cell comprising said gene and a polynucleotide encoding a dominant negative allele of a mismatch repair gene; and testing the cell to determine whether the cell harbors a mutation in said gene yielding at least one new biochemical feature of said antigen.
 36. The method of claim 35 wherein said new biochemical feature is selected from the group consisting of increased antigenicity and increased immunogenicity.
 37. The method of claim 35 wherein said testing comprises analyzing primary structure of said gene.
 38. The method of claim 35 wherein said testing comprises analyzing the secondary structure of said gene.
 39. The method of claim 35 wherein said testing comprises analyzing the antigenicity and immunogenicity of the polypeptide encoded by said gene.
 40. The method of claim 1 wherein said introduction of said polynucleotideis in the presence of at least one DNA mutagen.
 41. The method of claim 35 wherein said testing comprises analyzing a nucleotide sequence said gene.
 42. The method of claim 35 wherein said testing comprises analyzing mRNA transcribed from said gene.
 43. The method of claim 35 wherein said testing comprises analyzing the antigen protein encoded by the gene of interest.
 44. The method of claim 35 wherein said testing comprises analyzing the biochemical activity of the protein encoded by said gene.
 45. A hypermutable transgenic mammalian cell made by the method of claim
 35. 46. The transgenic mammalian cell of claim 45 wherein the cell is from primate.
 47. The transgenic mammalian cell of claim 45 wherein the cell is from rodent.
 48. The transgenic mammalian cell of claim 45 wherein the cell is from human.
 49. The transgenic mammalian cell of claim 45 wherein the cell is eucaryotic.
 50. The transgenic mammalian cell of claim 45 wherein the cell is prokaryotic
 51. A method for making randomly altered forms of a secreted antigen comprising introducing a polynucleotide encoding a tagged antigen into an MMR defective cell.
 52. The method of claim 51 wherein said tagged antigen is screened for increased antigenicity.
 53. The method of claim 51 wherein said tagged antigen is screened for increased immunogenicity.
 54. The method of claim 51 wherein the cells are made MMR defective by introducing at least one dominant negative allele of an MMR gene.
 55. The method of claim 51 wherein the cells are naturally MMR defective.
 56. A method of producing a mutated antigen in a reversibly unstable cell comprising introducing into a cell containing a preselected antigen of interest, an inducible expression vector comprising a polynucleotide encoding a dominant negative allele of a mismatch repair gene; inducing said cell to express said dominant negative mismatch repair gene; and detecting at least one new biochemical feature of said antigen.
 57. The method of claim 56 wherein said new biochemical feature is selected from the group consisting of a nucleotide mutation, increased antigenicity and increased immunogenicity.
 58. The method of claim 56 wherein said preselected antigen of interest is encoded on a polynucleotide previously transfected into said cell.
 59. The method of claim 56 further comprising ceasing induction of said dominant negative allele of a mismatch repair gene, thereby stabilizing said cell.
 60. The method of claim 58 further comprising isolating the polynucleotide previously transfected into said cell after detection of said new biochemical feature.
 61. An polynucleotide molecule for expressing an antigen in a hypermutable cell comprising an expression cassette wherein said cassette comprises a 3′ sequence encoding a plurality of histidine residues, a 5′ leader sequence of an expressed gene, and a polylinker to allow cloning of a nucleotide sequence encoding a preselected antigen.
 62. The polynucleotide molecule of claim 61 wherein said cell is a mammalian cell.
 63. The polynucleotide molecule of claim 61 wherein said cell is a human cell.
 64. The method of claim 63 wherein said 5′ leader sequence is a 5′ leader sequence of IL-2.
 65. A method of producing a mutated antigen comprising introducing a polynucleotide encoding a preselected antigen in the expression cassette said polynucleotide molecule of claim 61, and introducing said polynucleotide molecule into a cell comprising a dominant negative allele of a mismatch repair gene.
 66. A hypermutated antigen produced by the method of claim
 65. 67. A method of eliciting an immune response in an animal comprising administering to said animal an immunogenic amount of at least one hypermutated antigen of claim
 66. 68. The method of claim 67 wherein said antigen is a mutated form of an antigen derived from a pathogenic organism selected from the group consisting of, bacteria, fungi, protozoa, helminths, and viruses.
 69. An immunogenic composition comprising at least one hypermutated antigen of claim 66 and a pharmaceutically acceptable carrier. 